Rationale: The mobilization of bone marrow (BM) progenitor cells (PCs) is largely governed by interactions between stromal cell-derived factor (SDF)-1 and CXC chemokine receptor (CXCR)4. Ischemic injury disrupts the SDF-1-CXCR4 interaction and releases BM PCs into the peripheral circulation, where the mobilized cells are recruited to the injured tissue and contribute to vessel growth. BM PCs can also be mobilized by the pharmacological CXCR4 antagonist AMD3100, but the other components of the SDF-1-CXCR4 signaling pathway are largely unknown. c-kit, a membrane-bound tyrosine kinase and the receptor for stem cell factor, has also been shown to play a critical role in BM PC mobilization and ischemic tissue repair. Objective: To investigate the functional interaction between SDF-1-CXCR4 signaling and c-kit activity in BM PC mobilization. Methods and Results: AMD3100 administration failed to mobilize BM PCs in mice defective in c-kit kinase activity or in mice transplanted with BM cells that expressed a constitutively active c-kit mutant. Furthermore, BM levels of phosphorylated (phospho)-c-kit declined after AMD3100 administration and after CXCR4 deletion. In cells adhering to culture plates coated with vascular cell adhesion molecule 1, SDF-1 and stem cell factor increased phospho-c-kit levels, and AMD3100 treatment suppressed SDF-1-induced, but not SCF-induced, c-kit phosphorylation. SDF-1-induced c-kit phosphorylation also required the activation of Src nonreceptor tyrosine kinase: pretreatment of cells with a selective Src inhibitor blocked both c-kit phosphorylation and the interaction between c-kit and phospho-Src. Conclusions: These findings indicate that the regulation of BM PC trafficking by SDF-1 and CXCR4 is dependent on Src-mediated c-kit phosphorylation. (Circ Res. 2010;107:1083-1093.)