A dose-dependent requirement for E2A in the generation of DN3 cells from ETPs and DN2 cells. FACS-purified ETPs (250 cells/well) and DN2 cells (500 cells/well) from adult E2A^+/+, E2A^+/−, and E2A^−/− thymuses were cultured on OP9-DL1 for 10 days, and CD11b⁻CD8⁻ cells were analyzed for expression of (A) CD44 and CD25 or (B) NK1.1 and CD25. (C) Relative number of DN3 cells generated from E2A^+/+, E2A^+/−, or E2A^−/− ETPs and DN2s on day 10 of culture. Data represent the average of 5 independent experiments; *P < .001. (D) Single DN2 cells from E2A^+/+ and E2A^−/− mice were sorted into individual wells of a 96-well plate that contained OP9-DL1 and cultured for 10 days. Individual colonies were analyzed for expression of CD44 and CD25 on CD11b⁻CD8⁻ cells. Percentage of colonies that generated only DN3 cells (black) or a mixture of DN3 cells and other cell types (gray) or no DN3 cells (white) is shown. Numbers are combined from 3 independent experiments with 3 plates per sample. (E) Representative FACS plots of colonies generated from single E2A^+/+ and E2A^−/− DN2 cells.