Characterization of cell-autonomous T-cell developmental defects. (A) Lin⁻ thymocytes from transplant recipients were separated based on c-kit and CD25 expression into ETP (c-kit⁺ CD25⁻), DN2 (c-kit⁺ CD25⁺), and DN3 (c-kit⁻ CD25⁺) populations (top panels), and each population was analyzed for donor FL and BM-competitor–cell contribution in the 3 panels below. Two independent recipients of transplanted Cbfb^rss/−; FL cells are shown. Of the 9 transplant recipients of Cbfb^rss/−; FL cells, 6 resembled the pattern seen with rss/−; (1) and 3 resembled rss/−; (2). (B) CD4 expression is derepressed in Cbfb^rss/rss FL cells (bottom panels, arrows). (C) CD4 expression is up-regulated in the DN3 and DN4 populations. c-kit and CD25 were used to define the ETP, DN2, DN3, and DN4 populations from donor-derived Lin⁻ cells (scatterplots). Antibodies in the lineage-specific cocktail did not include CD4. Each population was analyzed for CD4 expression (histograms). (D) CD4 is not derepressed in Cbfb^rss/rss CD8 SP thymocytes. TCRβ^hi cells were analyzed for CD4 and CD8 expression in the panels below.