Mice lacking thymic function of the GTPase Rho show severe defects in fetal and adult thymopoiesis. Rho thymi are deficient in CD44+CD25+ pro-T cells and CD44-CD25+ early pre-T cells because Rho function is required for survival but not G1/S phase cell cycle progression in these populations. The selective apoptosis defect in Rho- prothymocytes can be rescued by expression of a bcl-2 transgene. A second function for Rho is seen in CD44- CD25- late pre-T cells: Rho regulates cell cycle progression but not survival of this population. These studies show that the critical processes of proliferation and survival are independently regulated during thymopoiesis and establish two different functions for Rho in the development of early thymic progenitors.