Karai's Vendetta - April first mentions her during her battle against Karai; distracted by the fact that April has lost her mother as well, April uses Karai letting her guard down to overpower her and manages to escape.
The Kraang Conspiracy - Mrs. O'Neil appears for the first time in an old family photo and Jack Kurtzman revealed the Kraang Hive Mind abducted her before April was born. The Hive Mind wanted, for yet unexplained reasons, a suitable human host for their plans of conquest for the Earth and finally found it in Mrs. O'Neil. They kidnapped and subjected her to experiments in which her human DNA was mixed with the genetic material of the Kraang. As a result of this engagement, April was born half Kraang, half human.
Buried Secrets - The turtles find April's long-lost mother frozen in a Kraang Stealth Ship buried under the farmhouse, though it's not really her, but rather a clone with Mrs. O'Neil's memories. Raphael called it "Mom Thing" as a nod to John Carpenter's "The Thing". It is revealed that the Kraang Hive Mind has been mutating April's ancestors for generations, including Mrs. O'Neil.
- In Buried Secrets, Mrs. O'Neil is voiced by Renae Jacobs, who voiced April in the 1987–1996 TV series.
- It was confirmed by executive producer Brandon Auman that Mrs. O'Neil is no longer alive.