Stromal cell-derived factor (SDF-1α), the ligand for CXCR4, is a chemokine that acts as a potent chemoattractant for hemopoietic progenitor cells. Stem cell factor/kit ligand (SCF/KL), an early acting cytokine, has recently been reported to enhance the chemotaxis induced by SDF-1α. However, very little is known about downstream signaling events following these receptor-ligand interactions. To investigate these events, we utilized a model progenitor cell line, CTS, which expresses both the CXCR4 and c-kit receptors. We observed strong Ca 2+ mobilization and enhancement of chemotaxis following treatment with SDF-1α or SCF/KL. A combination of these factors enhanced this chemotaxis in CTS cells as well as in CD34+ bone marrow cells. Prior treatment of CTS cells with pertussis toxin inhibited the SDF-1α-induced chemotaxis, suggesting that SDF-1α signaling involves a pertussis-sensitive G i-coupled protein. SDF-1α treatment resulted in a rapid phosphorylation of the focal adhesion molecules RAFTK (related adhesion focal tyrosine kinase), paxillin, and p130 cas, which then declined within minutes. SCF/KL alone or in combination with SDF-1α induced a rapid and sustained effect on phosphorylation of these substrates. SDF-1α treatment resulted in a rapid and robust activation of p44/42 mitogen-activated protein kinase compared with the relatively weak and delayed effect of SCF/KL treatment. Interestingly, a delayed but sustained activation of mitogen-activated protein kinase activation was observed when the factors were used in combination. Such cooperativity in downstream signaling pathways may explain the enhanced chemotaxis of progenitors observed with SDF-1α in combination with SCF/KL.