The stromal cell-derived factor-1 (SDF-1) is an alpha chemokine that binds to the CXCR4 receptor. Knock-out studies in mice demonstrate that this ligand-receptor pair is essential in hematopoiesis. One function of SDF-1 appears to be the regulation of migration of hematopoietic progenitor cells. We previously characterized signal transduction pathways induced by SDF-1 in human hematopoietic progenitors and found tyrosine phosphorylation of focal adhesion components, including the related adhesion focal tyrosine kinase (RAFTK), the adaptor molecule p130 Cas, and the cytoskeletal protein paxillin. To better understand the functional role of signaling molecules connecting the CXCR4 receptor to the process of hematopoietic migration, we studied SDF-1–mediated pathways in a model hematopoietic progenitor cell line (CTS), as well as in primary human bone marrow CD34⁺cells. We observed that several other focal adhesion components, including focal adhesion kinase (FAK) and the adaptor molecules Crk and Crk-L, are phosphorylated on SDF-1 stimulation. Using a series of specific small molecule inhibitors, both protein kinase C (PKC) and phosphoinositide-3 kinase (PI-3K) appeared to be required for SDF-1–mediated phosphorylation of focal adhesion proteins and the migration of both CTS and primary marrow CD34⁺ cells, whereas the mitogen-activated protein kinases ERK-1 and -2 were not. These studies further delineate the molecular pathways mediating hematopoietic progenitor migration and response to an essential chemokine, SDF-1.