Impaired pre-TCR–driven proliferation and accumulation of aberrant icTCRβ⁻ DN4 thymocytes in the absence of Rac1 and Rac2. (A) Flow cytometric analysis for incorporation of BrdU into DN3 and DN4 thymocytes from WT or Rac1^TRac2^−/− mice injected 4 hours earlier with BrdU. Markers indicate cells positive for BrdU, and thus were dividing during the labeling period. Numbers indicate percentage of cells falling into the marker. (B) Graph showing mean (± SEM) percentage of BrdU⁺ DN3 or DN4 thymocytes from mice of the indicated genotypes, analyzed as in panel A. (C) Histograms showing expression of intracellular CD3 (icCD3) in DN3 and DN4 thymocytes from WT or Rac1^TRac2^−/− mice (shaded plots) or stained with an isotype control antibody (open plots). (D) Histograms showing expression of intracellular TCRβ (icTCRβ) on DN3 and DN4 thymocytes from mice of the indicated genotypes. Markers indicate cells positive for icTCRβ. Numbers indicate percentage of cells falling into the markers. (E) Mean (± SEM) number of icTCRβ⁻ DN4 thymocytes from mice of the indicated genotypes. (F) Histograms showing expression of Thy1 on icTCRβ⁻ DN4 thymocytes and CD2 and CD5 on DN4 thymocytes from mice of the indicated genotypes. Statistically significant differences between Rac1^TRac2^−/− and WT mice are indicated (*P < .005; ***P < .001).