Transition failure from DN3a to DN3b is recapitulated in in vitro culture. Bone marrow progenitors (1 × 10⁴ cells) from 4- to 8-week-old adult BM were cultured with OP9-DLL1 cells supplemented with 5 ng/mL of Flt3L and 1 ng/mL of IL-7 for 18 days. Differentiated cells were harvested on the indicated days. (A) Representative dot plots showing the surface expression of CD4 and CD8 (top) from ATM^+/+ and ATM^−/− BM progenitors. (B) Percentages of CD4⁺CD8⁺ DP phase cells at the indicated time points. (C) Dot plots are gated on DN phase from ATM^+/+ and ATM^−/− BM progenitors. Red squares represent DN3 cells. Representative dot plots are shown when DN3 cells reached 1 × 10⁴ cells. Red dots indicate DN3b cells that are back-gated from the CD27⁺icTCR-β⁺ fraction. (D) Percentages of DN4 phase at the indicated time points. (E) Representative histograms showing expression of icTCR-β on DN3 cells. (F) Percentages of icTCR-β–positive cells on DN3 cells at the indicated time points. Data are representative of 3 independent experiments. Bar graphs represent mean ± SE. (G) Singly sorted DN3a and DN3b cells from each ATM^+/+ and ATM^−/− thymus were cultured on OP9-DLL1. Percentage of cells successfully differentiated to CD4⁺ and CD8⁺ (DP) phase were evaluated by flow cytometry on day 4. Bar graph represents mean percentage from 5 independent experiments. Data are mean ± SE. ***P < .001.