Rac1, one of the Rho family small guanosine triphosphatases, has been shown to work as a “molecular switch” in various signal transduction pathways. To assess the function of Rac1 in the differentiation process of CD4 single‐positive (CD4‐SP) T cells from CD4CD8 double‐positive (DP) cells, we used a DP cell line DPK, which can differentiate into CD4‐SP cells upon TCR stimulation in vitro. DPK expressing dominant‐negative (dn)Rac1 underwent massive apoptosis upon TCR stimulation and resulted in defective differentiation of CD4‐SP cells. Conversely, overexpression of dnRac2 did not affect differentiation. TCR‐dependent actin polymerization was inhibited, whereas early ERK activation was unaltered in dnRac1‐expressing DPK. We found that TCR‐dependent induction of Bcl‐2 was suppressed greatly in dnRac1‐expressing DPK, and this suppression was independent of actin rearrangement. Furthermore, introduction of exogenous Bcl‐2 inhibited TCR‐dependent induction of apoptosis and restored CD4‐SP generation in dnRac1‐expressing DPK without restoring TCR‐induced actin polymerization. Collectively, these data indicate that Rac1 is critical in differentiation of CD4‐SP from the DP cell line by preventing TCR‐induced apoptosis via Bcl‐2 up‐regulation.