The small Rho GTPases Rac1 and Rac2 have been implicated in regulating actin structures in a variety of cells, including hematopoietic stem cells and leucocytes. Actin oligomers are a significant structural component of the erythrocyte cytoskeleton. We explored the possible role of Rac1 and Rac2 signaling molecules in the dynamic assembly of actin in the red blood cells (RBC), and thus in the regulation of morphology and function of the erythrocyte cytoskeleton. Rac1 and Rac2 GTPases have been shown to have overlapping as well as distinct roles in actin organization, cell survival, and proliferation in hematopoietic stem cells (Gu et al. Science, 2003); we focused our study on the erythrocyte phenotype of Rac2−/− and Rac1−/−;Rac2−/− mice. Cre-recombinase-induced deletion of Rac1 genomic sequence was accomplished on a Rac2-null genetic background. Deletion of Rac1 after treatment with PolyI:PolyC to induce Cre recombinase was confirmed in bone marrow cells using DNA PCR and in erythrocytes by immunoblot. Since the erythrocytes consist a population of variable age, the optimal time of the maximum Rac1 deletion in erythrocytes was determined to be three to five weeks post induction. During this period, Rac1 protein in erythrocytes was decreased by 50–80% as determined by immunoblot densitometry. Rac2−/− and wild-type mice were subjected to the same treatment to control for any effects of PolyI:PolyC independent of the Rac1 deletion. Blood samples were obtained weekly after the completion of induction and the hematologic phenotype was studied by evaluation of complete blood counts, RBC indices, and reticulocyte counts. Erythrocyte morphology was examined on Wright-Giemsa smears of peripheral blood. Intact erythrocytes and erythrocyte ghosts were stained for actin with rhodamine-phalloidin and studied by confocal microscopy. The Rac2−/− mice appeared to have a rather mild erythrocyte phenotype with no significant anemia or reticulocytosis, although they did demonstrate a mild poikilocytosis and anisocytosis at baseline. The Rac1−/−;Rac2−/− mice developed a microcytic anemia with a hemoglobin drop of up to 30% in comparison to the baseline and to the wild-type hemoglobin values, with the nadir noted at three weeks post induction. The percentage of reticulocytes increased up to threefold in comparison to the control group. The mean corpuscular volume decreased up to 20% from the baseline in the Rac1−/−;Rac2−/− mice, and remained decreased up to six weeks post induction with an elevated red blood cell distribution width. Significant anisocytosis and poikilocytosis were observed with fragmented erythrocytes in the peripheral blood smear. Filamentous actin in the RBC cytoskeleton stained with rhodamine-phalloidin appeared to have a uniform distribution in intact and ghost erythrocytes under confocal microscopy. However, Rac1−/−;Rac2−/− erythrocytes demonstrated punctuate lesions on the cell surface while Rac1−/−;Rac2−/− erythrocyte ghosts appeared to collapse into irregular shapes. These data suggest that deficiency of Rac1 and Rac2 GTPases in mice cause a microcytic hemolytic anemia with poikilocytosis and red cell fragmentation indicating a possible dynamic regulation of the erythrocyte cytoskeleton organization by these signaling molecules.