Adult hematopoietic progenitor cells (HPCs) are maintained by highly coordinated signals in the bone marrow. The molecular mechanisms linking intracellular signaling network of HPCs with their microenvironment remain poorly defined. The Rho family GTPase Rac1/Rac2 has previously been implicated in cell functions involved in HPC maintenance, including adhesion, migration, homing, and mobilization. In the present studies we have identified R-Ras, a member of the Ras family, as a key signal mediator required for Rac1/Rac2 activation. We found that whereas Rac1 activity is up-regulated upon stem cell factor, integrin, or CXCL12 stimulation, R-Ras activity is inversely up-regulated. Expression of a constitutively active R-Ras mutant resulted in down-regulation of Rac1-activity whereas deletion of R-Ras led to an increase in Rac1/Rac2 activity and signaling. R-Ras(-/-) HPCs displayed a constitutively assembled cortical actin structure and showed increased directional migration. Rac1/Rac2 inhibition reversed the migration phenotype of R-Ras(-/-) HPCs, similar to that by expressing an R-Ras active mutant. Furthermore, R-Ras(-/-) mice showed enhanced responsiveness to G-CSF for HPC mobilization and exhibited decreased bone marrow homing. Transplantation experiments indicate that the R-Ras deficiency-induced HPC mobilization is a HPC intrinsic property. These results indicate that R-Ras is a critical regulator of Rac signaling required for HPC migration, homing, and mobilization.