Therapeutic mobilisation of bone marrow progenitor cells (BM PCs) is a novel strategy for cardiovascular repair. Both CXCR4 antagonism and c-kit blockade can rapidly and potently mobilise BM PCs; however, the functional interaction between CXCR4 and c-kit remains unclear. We treated c-kit-deficient (c-kit W/W-V) and wild-type (WT) mice with CXCR4 antagonist AMD3100 and evaluated PCs in the peripheral blood (PB) with a colony-forming assay. AMD3100 treatment for 2 h dramatically increased the number of PCs in the PB in WT mice but not in c-kit W/W-V mice (c-kitW/W-V vs WT: 30 vs 194 colonies/ml blood, p<0.01). To confirm that c-kit deficiency impairs BM PC mobilisation by AMD3100, we developed an in vivo BM niche clearance/occupation assay. The c-kit W/W-V and WT mice were firstly treated with AMD3100 to remove PCs from the niche, and 2 h later, transplanted with eGFP transgenic BM cells to competitively occupy the niche. After 3 h, BM cells were isolated and analysed by FACS. Despite the total donor-derived (eGFP+) cells were similar between WT and c-kit W/W-V recipients, the donor-derived CXCR4-expressing PCs, including eGFP+CXCR4+Lin-, eGFP+CXCR4+Lin-Sca1+, and eGFP+CXCR4+Lin-ckit+ cells, were much fewer in the c-kit W/W-V mice (c-kitW/W-V vs WT: 19.7% vs 30.6%, p<0.01; 20.3% vs 29.1%, p<0.05; and 6.7% vs 17.9%, p<0.001, respectively), indicating that c-kit deficiency specifically reduced the capacity of AMD3100 to clear the CXCR4+ PC niche. To better understand the mechanisms, we designed an ex vivo adhesion assay. Mouse BM mononuclear cells were isolated and applied onto plates pre-coated with BM stromal protein VCAM-1, followed by addition of AMD3100. The adhesion of BM cells to VCAM-1 resulted in marked c-kit phosphorylation. Interestingly, AMD3100 significantly attenuated the c-kit phosphorylation. In vivo, AMD3100 treatment for 15 min significantly reduced the level of phospho-c-kit in the BM as assessed by Western blotting of the BM lysates. Consistently, immunofluorescence staining of BM niche demonstrated a significantly lower ratio of phospho-ckit+/total ckit+ PCs in AMD3100-treated mice as compared to PBS-treated mice. We conclude that c-kit plays a critical role in CXCR4-mediated BM PC niche maintenance and mobilisation.