CXCR4 receptor expression is required for the retention of granulocyte precursors and mature neutrophils within the bone marrow, and disruption of the SDF-1/CXCR4 axis in the bone marrow results in the mobilization of myeloid lineage cells to the peripheral circulation. We report that G-CSF down-regulates CXCR4 expression in bone marrow–derived murine and human myeloid lineage cells. When exposed to G-CSF, murine Gr1⁺ bone marrow myeloid cells display a time-dependent reduction of cell-surface CXCR4 and respond poorly to SDF-1 in attachment and migration assays. Bone marrow–derived cells of nonmyeloid lineage display no change in surface CXCR4 expression upon exposure to G-CSF. Compared with controls, mice treated with G-CSF for mobilization of hematopoietic progenitor cells display reduced levels of CXCR4 selectively in bone marrow Gr1⁺ myeloid cells. Since bone marrow myeloid cells express G-CSF receptors and G-CSF rapidly reduces CXCR4 expression in purified Gr1⁺ cells populations, these results provide evidence that G-CSF acts directly on myeloid lineage cells to reduce CXCR4 expression. By down-regulating CXCR4 expression in bone marrow myeloid cells and attenuating their responsiveness to SDF-1, G-CSF promotes their mobilization from the bone marrow to the peripheral blood.