Despite Fuyutsuki's misgivings, Yui served as the test subject for the Contact Experiment of Unit 01 in 2004, saying that "everything happens as it must." She invited her son Shinji to Gehirn to watch what would be her final moments: Yui "disappeared" during testing — leaving no body behind — and was proclaimed dead that same year after efforts to recover her failed. Although Gehirn considered it an accident, it is arguably clear that what happened was Yui's intention. Living on as the soul of the Eva, she continues to ensure a "bright future" for Shinji and the rest of humanity, seeing to it personally that Seele's scenario is never entirely fulfilled.
Yui appears briefly in episode 16 as a pair of glowing arms which reach out to Shinji as he slips into unconsciousness in Unit 01's entry plug; the Eva tears its way out of Leliel's shadow immediately thereafter. In episode 20, while Shinji is absorbed in Unit 01, he sees a scene from the past: Yui and Gendo are under a tree, and Yui is breastfeeding Shinji. Yui and Gendo talk, and Yui sees and talks briefly with Shinji then urges him to leave the Eva and return to the world.
Yui's background is not covered in the series, but according to information provided in the Neon Genesis Evangelion 2 PS2 game, she is the daughter of an influential member of Seele, which explains why she had the backing of this powerful organization. However, the canonocity of this has never been officially stated.
Shinji is equally affected by his mother's absence, as that and Gendo's subsequent abandonment of him is a massive trauma that he has carried his entire life and is what has led to his avoidant, non-confrontational nature. At the same time, her maternal protectiveness of Shinji as the soul of Unit 01 is solely responsible for the Eva being the formidable fighter it is.
Although Fuyutsuki was never more than a mentor and friend to Yui, it is strongly implied that he was in love with her to some degree, as he was shocked to learn that she was marrying Gendo (whom he disliked at the time); also, in The End of Evangelion Lilith/Rei takes the form of Yui to achieve Fuyutsuki's Instrumentality.
In Yoshiyuki Sadamoto's manga adaptation, Yui's role is identical to that in the anime, but is expanded slightly. In the aftermath of the fight with the Angel Zeruel, she appears inside Unit 01 and tried to convince Shinji to remain with her. Initially, it seems to be working well: Shinji refuses to come out when attempts are made to retrieve him, forcing the contents of the Entry Plug to spill out. However, Rei mentally calls out to Shinji, and Yui's face peels away to reveal that it is Unit 01 itself who has disguised itself as Yui to try to trap Shinji inside itself. Upon seeing Unit 01's true self, a horrified Shinji struggles with the Eva, crying out to Yui. A female form appears behind the disfigured body of Unit 01, and appears to merge with Shinji. Later, Shinji sees a scene from the past: His parents were under a tree, and Yui was breastfeeding an infant Shinji. After a short conversation with Gendo, Yui then notices her grown son and asks him to join her. Surprisingly, Shinji is unable to move, despite his desire to get close to his mother. Yui then convinces Shinji to leave the confines of Unit 01 and go back to the world.
While the manga is in black and white, the cover of volume 8 depicts Yui with reddish-blonde hair and light green eyes, rather than the brown hair and dark green eyes she has in the anime.
She is shown to be a jovial mother and hardworking scientist, although she does not tolerate too much foolishness. (When Gendo shone his torch in a scary fashion during a blackout, Yui gave him a comical smack.)