RhoA is a founding member of Rho GTPase family and is a well-recognized regulator of actin cytoskeleton dynamics. Signal pathways transduced by RhoA are involved in cell migration, polarity, morphogenesis, proliferation, survival, and cell fate decision. Conditional gene targeting of Rhoa in murine blood system induces acute hematopoietic failure due to the loss of multilineage hematopoietic progenitor cells (HPC) caused by a cytokinesis defect and necrosis. Here we describe a method to conditionally induce Rhoa gene knockout in murine blood cells and a rescue by exogenous RhoA expression with lentivirus in HPCs, an approach that has general applicability in studying in vivo function of Rho GTPases and their regulators/effectors by gene targeting.