Patients with myelodysplasia (MDS) show a differentiation defect in the multipotent stem-cell compartment. An important factor in stem-cell differentiation is their proper localization within the bone marrow microenvironment, which is regulated by stromal cell-derived factor (SDF-1). We now show that SDF-1-induced migration of CD34(+) progenitor cells from MDS patients is severely impaired. In addition, these cells show a reduced capacity to polymerize F-actin in response to SDF-1. We demonstrate a major role for Rac and phosphatidylinositol 3-kinase (PI3K) and a minor role for the extracellular signal-regulated kinase (ERK)1/2 signaling pathway in SDF-1-induced migration of normal CD34(+) cells. Furthermore, SDF-1-stimulated activation of Rac and the PI3K target protein kinase B is impaired in CD34(+) cells from MDS patients. Lentiviral transduction of MDS CD34(+) cells with constitutive active Rac1V12 results in a partial restoration of F-actin polymerization in response to SDF-1. In addition, expression of constitutive active Rac increases the motility of MDS CD34(+) cells in the absence of SDF-1, although the directional migration of cells toward this chemoattractant is not affected. Taken together, our results show a reduced migration of MDS CD34(+) cells toward SDF-1, as a result of impaired activation of the PI3K and Rac pathways and a decreased F-actin polymerization.