Rho GTPases including RhoA, Rac1, and Cdc42 are a class of intracellular signaling proteins critical for the regulation of cytoskeleton organization, adhesion, and migration. Molecular mechanisms of mammalian cell migration were first revealed in fibroblasts where RhoA, Rac1, and Cdc42 facilitate in the multistep process including establishment and maintenance of polarity, formation of actin-rich protrusions, remodeling of adhesive contacts, and generation of force. In hematopoietic stem/progenitor cells, Rho GTPases relay signals from chemokines and cytokines such as SDF-1α and SCF to the actin and microtubule cytoskeleton through effector kinases and/or adaptor molecules that affect adhesion or transcription. Comprehensive use of murine conditional gene knockout technology combined with biochemical approaches in recent studies allows for physiologically relevant investigations of the involvement of Rho GTPases in hematopoietic stem/progenitor cell migration, providing important mechanisms for the stem/progenitor maintenance.