The Rho family of guanosine triphosphatases (GTPases) is composed of members of the Ras superfamily of proteins. They are GTP‐bound molecules with a modest intrinsic GTPase activity that can be accelerated upon activation/localization of specialized guanine nucleotide exchange factors. Members of this family act as molecular switches and are required for coordinated cytoskeletal rearrangements that are crucial in a set of specialized functions of mammalian stem cells. These functions include self‐renewal, adhesion, and migration. Mouse gene‐targeting studies have provided convincing evidence of the indispensable and dispensable roles of individual members of the Rho GTPase family and the putative upstream and downstream mediators in stem cell‐specific functions. The role of Rho GTPases and related signaling pathways previously seen in other cell types and organisms have been confirmed in mammalian hematopoietic stem cells (HSCs), and new signaling pathways and unexpected functions unique to HSCs have been identified and dissected. This review summarizes our current understanding of the role of Rho family of GTPases on HSC and progenitor activity through cytoskeleton‐mediated signaling pathways, providing insight about relevant signaling pathways that regulate mammalian stem cell self‐renewal, adhesion, and migration.