She and her father, Kirby O'Neil, first met the turtles when the Kraang came to kidnap her. Donatello fell in love with her the moment he met her and called her the most beautiful girl he's ever seen. Raphael tells him she's the only girl he's ever seen. Though she was afraid of the turtles at first, Donatello is able to get her to trust them. When the Kraang kidnap her, the turtles go to rescue her and her father. While they were able to rescue April, Kirby remained in the hands of the Kraang. She moved into her aunt's house and was welcomed to hang out at the turtles' lair.
Though she is unaware of Donatello's crush on her, she has the closest relationship with Donatello over the other turtles. In Monkey Brains, it's revealed she was an empath, revealing a mutant mokey was actually a human scientist. At the end of the episode, Splinter offers to train her to be a kunoich, a female ninja. In later episodes, she is shown to have some spying and combat skills, though not on par with the turtles. She used her spying skills to even spy on the Shredder, even though the turtles and Splinter were against it at first. She is also a skilled hacker and was able to download the blueprints to the TCRI building. In the episode TCRI, it is revealed that the Kraang were after her, not her father. The reason why is unknown. However, in Karai's Vendetta, the Shredder learns of April's connection to the Kraang and the turtles and she is now a target of the Shredder as he believes she can be useful bait to the turtles. Karai goes after April, but in the end April is able to win the fight, though because she is now a target to both the Kraang and the Shredder, she has to stay in the turtle's lair to be safe.
It has been confirmed Casey Jones will be in the second season. Since April was Casey's love interest in the 2003 series and the comics, it is possible he will be Donatello's rival over April.
This is the youngest incarnation of April O'Neil to date.
Her father is a psychologist.
In Karai's Vendetta, it is shown that on her phone, Donatello's profile shows him surrounded by flowers and the title, "Donny Boy." This hints that she may return Donatello's feelings.