The mechanisms that regulate hematopoietic progenitor cell (HPC) mobilization from the bone marrow to blood have not yet been defined. HPC mobilization by granulocyte colony-stimulating factor (G-CSF), cyclophosphamide (CY), or interleukin-8 but not flt-3 ligand is markedly impaired in G-CSF receptor–deficient (G-CSFR–deficient) mice. G-CSFR is expressed on mature hematopoietic cells, HPCs, and stromal cells, which suggests that G-CSFR signals in one or more of these cell types was required for mobilization by these agents. To define the cell type(s) responsible for G-CSF–dependent mobilization, a series of chimeric mice were generated using bone marrow transplantation. Mobilization studies in these chimeras demonstrated that expression of the G-CSFR on transplantable hematopoietic cells but not stromal cells is required for CY- or G-CSF–induced mobilization. Moreover, in irradiated mice reconstituted with both wild type and G-CSFR–deficient bone marrow cells, treatment with CY or G-CSF resulted in the equal mobilization of both types of HPCs. This result held true for a broad spectrum of HPCs including colony-forming cells, CD34⁺lineage⁻ and Sca⁺ lineage⁻cells, and long-term culture initiating cells. Collectively, these data provide the first definitive evidence that expression of the G-CSFR on HPCs is not required for their mobilization by G-CSF and suggest a model in which G-CSFR–dependent signals act in trans to mobilize HPCs from the bone marrow.