Deletion of 1 Gata3 allele restores the ability of E2A^−/− DN2 thymocytes to differentiate to the DN3 stage. (A) FACS analysis for BrdU in DN2 or DN3 cells from control, E2A^−/−, and E2A^−/−Gata3^+/Δ mice determined 24 hours after in vivo administration of BrdU. Shaded histogram is negative control. Representative FACS plots from 3 independent experiments are shown. (B) Limiting dilution analysis of DN3 cell development from control, E2A^−/−, and E2A^−/−Gata3^+/Δ DN2 cells cultured on OP9-DL1 for 12 days. Control DN2 cells were plated at 3, 9, and 27 cells/well; E2A^−/− and E2A^−/−Gata3^+/Δ DN2 cells were plated at 5, 15, and 45 cells/well with 48 replicate cultures for each cell concentration. On day 12, each well was scored for growth, followed by FACS analysis for surface expression of CD44 and CD25. Results are plotted as percentage of negative cultures versus input cell number. The frequency of responding cells was determined as the input cell concentration where 37% of wells are negative for DN3 cells. (C) Representative FACS plots from single wells seeded with DN2 cells of the indicated genotype. (D) Percentage of DN3 cells in each positive well (with the highest number of cells plated) of control, E2A^−/−, and E2A^−/−Gata3^Δ/+ DN2s culture; *P < .05.