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Lilith was a [[Demon|white-eyed demon]]<ref name="three12">[[3.12 Jus in Bello]]</ref> who was at one point human. She is the first demon to ever be created, her [[soul]] having been twisted by [[Lucifer]] as an act of spite against [[God]] that got him expelled from [[Heaven]].<ref name="four21">[[4.21 When the Levee Breaks]]</ref> As such, she preceded both the [[Princes of Hell]] and the [[Knights of Hell]].<ref name="twelve13">[[12.13 Family Feud]]</ref> Due to her status as the first demon, she is the last of [[the 66 Seals]], with her death breaking the last seal and releasing Lucifer from the [[Lucifer's Cage|the Cage]] he was imprisoned in for her creation.<ref name="four22">[[4.22 Lucifer Rising]]</ref>

Lilith is was later revealed to be among the hundred or more [[demons]] seen escaping [[Hell]] through the open [[Devil's Gate]] in this episode; like the other demons, she appears appeared as [[demon smoke]].

When [[Sam]] and [[Dean]] are captured by the FBI and held at a local police station, they find themselves under siege by a horde of [[demons]]. When [[Ruby]] shows up to help, she explains that the demons are trying to kill Sam at Lilith's command, revealing that Lilith has succeeded in taking over as the new leader of [[Azazel]]'s demon army. Ruby is the first character to name Lilith.

At the end of the episode, [[BelaTalbot]] reveals revealed that Lilith told her about Dean's [[Deandeal]]'s deal and that Lilith she had claimed to hold the contracts to every [[deal]], including Dean's and Bela's. She also reveals revealed that she had stolen the [[the Colt]] to bargain with Lilith but that it hadn't been enough; Lilith wanted her to kill [[Sam]], too. Bela tells Dean all of this in the hope that Dean will be able to kill Lilith.

[[Sam]] and [[Dean]] plan to kill Lilith to save Dean from his [[deal]]. With [[Bobby]]'s assistance, they discover that she is in New Harmony, Indiana, on what [[Ruby]] refers to as "shore leave." Lilith has [[Possession|possessed]] another little girl, killed the girl's "mean old babysitter" and left the elderly woman's corpse to rot in the hallway, and is terrorizing the girl's family (the Fremonts) while her [[demon]]s demons possess the neighborhood to keep watch. After killing a pet named Freckles for being "mean" to her, Lilith walks in on the Fremonts discussing whether or not they should try to try to escape. She asks what they were talking about, but they hastily change the subject. Her [[meatsuit]]'s father asks if she will let them go and her demeanor become icy—she wants to know why they want to leave, and suggests menacingly that they don’t love her anymore and that they shouldn't upset her or she will kill them. After a pause, she cheerfully accepts their apology and asks the father to push her on the swings outside. She later has them celebrate her birthday again (it is implied that she has been making them celebrate it everyday). She casually asks the grandfather why he asked a neighbor for help earlier. She calls him a liar when he feigns innocence, then turns her attention to the parents and demands to know if they knew. They lie that they didn't and when the grandfather pleads plead for help, Lilith snaps snaped his neck with [[telekinesis]] before warning the terrified parents not to scream because it'll irritate her. Later that night, she forces Lilith forced the mother to curl up in bed with her and read her the same story over and over again.

Sam, Dean, and Ruby enter the Fremont household after Bobby has barred Lilith's demons from coming to her aid by setting the water sprinklers to go off with [[holy water]]. Sam finds the girl asleep with her mother awake beside her. The mother begs Sam to kill her daughter as Sam approaches, but right as Sam is about to kill the girl with [[Ruby's knife]], Dean calls him off; as he temporarily has the ability to see the true faces of demons, he knows Lilith is no longer possessing the little girl. Sam entreats Ruby to teach him to use his powers to kill Lilith and save Dean, but she tells him that it's too late to do that now. As a [[hellhound]] comes for Dean, the trio barricade themselves in the house's office. When Ruby is trying to convince Sam to give her her the knife back so that she can try to use it to fend off the hellhound, Dean suddenly notices the difference in the demonRuby's true demon face from how it looked earlier, and realizes that the woman previously possessed by Ruby is now possessed by Lilith. She uses telekinesis to pin Lilith then pinned Sam to a wall and Dean to a table, and indicates indicated that she sent Ruby to [[Hell]] before taking the other demon's meatsuit for herself. Remarking that she likes possessing the woman because "it's all grown-up and pretty," she forces a kiss on Sam before she rejects his attempt to trade his life for Dean's, taunting him that he doesn't have anything that she wants. She ignores Dean's desperate attempt to buy time by getting her to talk, instead setting the hellhound on him and watching on with amusement as it mauls Dean to death. Lilith turns her power on Sam, but it has no effect and she quickly vacates her meatsuit when Sam goes to attack her with Ruby's knife.

[[Dean]] asks [[Sam]] how he had survived when Lilith had been trying to kill him; Sam he tells him Dean how she tried to kill him with her light and had fled when he was immune to it. Later, [[Ruby]] tells Sam that not even Lilith can could resurrect a human in [[Hell]].

When arguing about whether or not [[Castiel]] is a real [[angel]], [[Sam]] points out that even Lilith is scared of [[Ruby's knife]]. [[Henriksen]]'s [[ghost]] reveals that instead of killing everyone in the police station in [[3.12 Jus in Bello]] right away, Lilith had entertained herself by torturing them all for forty-five minutes first, skinning [[Nancy]] alive and saving Henriksen for last. Towards the end of the episode, Castiel tells [[Dean]] that it was Lilith who raised the [[Witnesses]], and explains that she had done so to break one of [[the 66 Seals]]; when the last one is broken, [[Lucifer]] will be freed. Castiel attributes her choice in Witnesses to her twisted sense of humor.

At the start of the episode, [[Sam]] and [[Ruby]] are interrogating a [[demon]] they have captured for Lilith's whereabouts, but the demon defiantly refuses refused to tell them anything.

When infected with [[ghost sickness]], [[Dean]] has a hallucination of Lilith in the form of the Fremont girl, who is happy to see him, having "missed" him. She reveals that four months is equivalent to forty years in [[Hell]] and that Dean remembers every moment of it. She admits that she is not real, but says that it doesn't matter because Dean is still going to die and go back to [[Hell]]. She tells Dean to listen to his heart and screams "ba-boom!" in rhythm with his heartbeats, making his heart beat faster and faster, but Dean is cured of ghost sickness before he can die of a heart attack.

When [[Dean]] asks [[Sam]] what he'd wish for, if he could have anything, Sam says that he'd wish for "Lilith's head on a plate, bloody" instead of the normal life he used to want.

In flashbacks set after [[3.16 No Rest for the Wicked]] but before [[4.01 Lazarus Rising]]Months prior, [[Ruby]] claims that Lilith tortured her in [[Hell]] as punishment for helping [[Sam]]. Ruby endures , and endured Lilith's "creative" torments and tries to desperately to convince Lilith that she is repentant and can now be trusted; eventually, Lilith gives gave Ruby the opportunity to escape further torture by finding and killing Sam for her. Ruby agrees, and Lilith sets set her free and sends her back to Earth. Instead of fulfilling her part of the deal once she found Sam, however, Ruby again betrays Lilith by teaching Sam to use his powers so that he can kill Lilith and avenge [[Dean]]. A month after they begin began training, Sam finds omens of Lilith's presence and decides to go after her despite Ruby telling him that he isn't ready to fight Lilith yet. He walks into a trap Lilith had set up, but Lilith herself is not there—the [[demon]]s demons who ambush Sam tell him that she couldn't make it, though she wanted to. Lilith's plan is then foiled by Ruby saving Sam.

In the present, [[AnnaMilton]] tries to explain to her psychiatrist that Lilith is breaking [[the 66 Seals]], revealing that the [[angel]]s angels cannot stop Lilith because there are so many potential seals to defend and no one knows which ones Lilith will target next. When Sam and Dean visit the mental hospital, they find that one of Anna's sketches depicts Lilith (using another little girl for a [[meatsuit]]) raising the [[Witnesses]] from [[4.02 Are You There, God? It's Me, Dean Winchester]].

[[Ruby]] tells [[Sam]] that Lilith has had already broken thirty-four of [[the 66 Seals]]. As Sam is the only one who can kill Lilith, Ruby urges Sam to focus his efforts on finding and killing Lilith to "cut the head off the snake" and stop the seal-breaking. Although Sam is still set on killing Lilith, however, he doesn't want to use what Ruby says is the only way he can do it (later revealed to be drinking [[demon blood]]). At the end of the episode, he but finally gives in.

[[Ruby]] is working on tracking Lilith down and has arranged for [[Sam]] and [[Dean]] to meet up with her to follow through on some leads; they are traveling to get to their meeting spot when [[Castiel]] and [[Uriel]] kidnap Dean to interrogate [[Alastair]] for them. Alastair reveals that the reason Lilith had wanted Dean in [[Hell]] in the first place was so that he could break the first of [[The the 66 Seals]] by torturing other [[souls]]. Alastair also reveals revealed that Lilith is not behind the [[angel]] murdersdeaths; telling him if she were able to kill angels, she would've killed a thousand instead of just seven.

[[ChuckShurley]] warns [[Sam]] and [[Dean]] that Lilith is coming, and predicts that she and Sam will have sex. Dean wants to leave immediately to avoid Lilith, but Sam insists on confronting her. She Lilith eventually appears appeared to him when he is Sam was alone in his and Dean's motel room. Instead of her usual little girl [[meatsuit]]s, she is [[Possession|possessing]] Lilith chose to possess a dental hygienist from Bloomington, Indiana. She breaks the [[devil's trap]] Sam laid down to capture her by touching it and burning a portion of it away, and when he Sam tries to kill her with his powers, she is barely affected, commenting that he is "strong, but not that strong." Likewise, she is unable to kill him with her powers.

Lilith reveals that she has come to make a [[deal]] with Sam; she will stop breaking [[The the 66 Seals]], but only in return for Sam and Dean's lives. When Sam questions why Lilith would want to stop breaking seals, she reveals that she is destined to die right before the [[Apocalypse]] is set in motion. Sam still hesitates and Lilith calls him selfish for valuing his life over those of six billion innocent people, before suggesting that his selfishness comes from the [[Demon Blood]] demon blood in him, goading Sam into agreeing to her deal. She beckons him to the bed to seal the deal with sex, but as he lies down and embraces her, he grabs [[Ruby's knife]] in order to kill her. She Lilith overpowers him and seizes the knife, but before she can do anything else, Dean arrives with Chuck in tow. Dean warns her to leave before the [[archangel]] tasked with protecting Chuck (possibly [[Raphael]]) comes and kills her. Though reluctant to leave her business with Sam unfinished, Lilith heeds Dean's words and vacating the body she is possessing, leaving the dental hygienist motionless on the floor. When Dean asks Sam whether he would've taken the deal, Sam denies it, as he suspects that Lilith would've found some way to "weasel out of it." He does note that she's scared of something and that she was right about dying before the Apocalypse, because he plans to make sure of it.

[[Sam]] tells [[Dean]] that he is drinking [[Demon Blood]] demon blood to get strong enough to kill Lilith, but Dean says tells him that he and [[Bobby]] will kill her instead while they keep Sam locked in [[Bobby's Panic Room]]. Later, [[Ruby]] reveals that Lilith is "[[Lucifer]]'s first"—the — the first [[demon]] created from a humansoul, and the only one who can break the last of [[the 66 Seals]]. She also reveals revealed that Lilith has a "personal chef" who kidnaps babies for her to eat and is currently [[Possession|possessing]] [[Cindy McClellan]]. Sam and Ruby decide to capture the [[demon]] in order to interrogate her for Lilith's whereabouts so that they can kill Lilith before she breaks the last [[the 66 Seals|seal]].

In a flashback to 1972, [[Azazel]] communicates with [[Lucifer]] after sacrificing six nuns in a chapel in [[St. Mary's Convent]] in Ilchester, Maryland, [[The Nun|the seventh nun ]] serving as Lucifer's mouthpiece. Lucifer tells Azazel that Lilith is needed for him to be released, and when Azazel notes that Lilith is trapped deep in [[Hell]], Lucifer tells Azazel how he can free her, starting with instructions to find "[[Sam|a very special child]]." This scene implies (and [[Eric Kripke]]'s commentary on the episode confirms) that Azazel had had the [[Devil's Gate]] opened in [[2.22 All Hell Breaks Loose: Part Two]] for the sole purpose of freeing Lilith so that she could break [[The 66 Seals]].

In the present, the [[demon]] [[Possession|possessing]] [[Cindy McClellan]] is reluctant to give Sam and [[Ruby]] information on Lilith for fear that Lilith will kill her. When Sam tortures her, however, she reveals that Lilith will be at St. Mary's Convent the following night to break the last of seals, though this is all that she knows regarding the last seal. Lilith once again possesses the dental hygienist, and she and her minions set up the destruction of the final seal at St. Mary's Convent. Sam and Ruby later arrive at the convent, kill Lilith's minions, and attack Lilith herself, with Sam pinning her to the altar with [[telekinesis]] while trying to kill her with his powers, to no avail. Meanwhile, [[Dean]] learns that the [[angel]]s angels have been allowing Lilith to break the seals because they want the [[Apocalypse]] to happen for their own ends, and that Lilith's death itself is the final seal that, should it come to pass, will release Lucifer. [[Castiel]] has a change of heart and sends Dean to stop Sam from killing Lilith, but Ruby bars Dean from entering the chapel. Lilith laughs at Sam's hesitation at the sound of Dean's voice, and taunts him about turning himself into a monster by drinking [[Demon Blood]]demon blood, which pushes Sam to finally finish her Lilith off, thus breaking the last seal. Blood issues from her dead [[meatsuit]]'s mouth and forms the entrance to [[Lucifer's Cage]] which Lucifer then rises from.  Ruby then triumphantly reveals to Sam that she has had secretly been working as Lilith's double-agent all along and that it had been on Lilith's orders that she manipulated him and tricked him into breaking the last seal, explaining that Lilith and Azazel had done everything they did in order to get Sam to this point. Furthermore, Ruby reveals that Lilith had been the only one who knew her true loyalty.

At the end of the episode, Lilith is mentioned by [[Dean]] when he admits that, although he still doesn't approve of [[Sam]] having drank [[Demon Blooddemon blood]] to do it, he is glad that Sam managed to kill her and he knows that Sam hadn't known at the time that her death was the last [[The 66 Seals|seal]]. Sam reminds him that his killing her had started the [[Apocalypse]], but Dean points out that neither of them could have foreseen that killing Lilith would turn out to be a bad thing.

Lilith is mentioned by [[BeckyRosen]], an avid fan of [[the Supernatural Books]]. Becky discloses to [[Sam]] that, contrary to what they had believed, [[Bela]] had given [[the Colt]] to Lilith's "right-hand man," [[Crowley]], not Lilith herself. Becky has the impression that, as well as her servant, Crowley was Lilith's lover.

In a flashback set four years before the episode2008, [[Sam]] and [[Dean]] are trying to track Lilith down. They interrogate a [[Jeffery!Demon|demon]] [[Possession|possessing]] [[Jeffrey]] and he eventually reveals that she has a lieutenant, Merrick, who is stationed in New Orleans, Louisiana. For giving the Winchesters this information, the demon is imprisoned deep in [[Hell]] after Sam and Dean [[Exorcism|exorcise]] him.

After Bobby and [[Rufus]] trap the a [[soul eater]] in its nest in Grand Rapids, Michigan, Dean calls Bobby about a possible lead on Lilith in Maine.