Impaired thymic development and accumulation of aberrant TCRβ⁻ DN4 thymocytes in the absence of Rac1 and Rac2 is intrinsic to the T-cell lineage. Mixed radiation chimeras were made using bone marrow from either Rac1^+/+Rac2^+/+hCD2-iCre (WT) or Rac1^flox/floxRac2^−/−hCD2-iCre (Rac1^TRac2^−/−) mice (both Ly5.2⁺) and bone marrow from B6.SJL mice (Ly5.1⁺). (A) Graph showing the mean (± SEM) ratio between Ly5.2⁺ and Ly5.1⁺ cells in each thymic developmental compartment, defined as in Figure 1, with the addition of intermediate single-positive (ISP) cells defined as CD4⁻CD8⁺TCRβ⁻. These are cells in transit between the DN4 and DP compartments. The ratios were all normalized to the ratio of Ly5.2⁺ to Ly5.1⁺ cells in the DN2/3 compartment, which was set to 1. (B) Graph showing mean (± SEM) percentage of cells that were either positive or negative for intracellular TCRβ (icTCRβ) in the Ly5.2⁺ DN3 or DN4 compartments of the mixed radiation chimeras. Colors indicate genotypes as in panel A. Statistically significant differences between Rac1^TRac2^−/− and WT chimeras are indicated (*P < .01; **P < .001).