Stem cell homing into the bone microenvironment is the first step in the initiation of marrow-derived blood cells. It is reported that human severe combined immunodeficient (SCID) repopulating cells home and accumulate rapidly, within a few hours, in the bone marrow and spleen of immunodeficient mice previously conditioned with total body irradiation. Primitive CD34⁺CD38^−/lowCXCR4⁺ cells capable of engrafting primary and secondary recipient mice selectively homed to the bone marrow and spleen, whereas CD34⁻CD38^−/lowLin⁻ cells were not detected. Moreover, whereas freshly isolated CD34⁺CD38^+/high cells did not home, in vivo stimulation with granulocyte colony-stimulating factor as part of the mobilization process, or in vitro stem cell factor stimulation for 2 to 4 days, potentiated the homing capabilities of cytokine-stimulated CD34⁺CD38⁺ cells. Homing of enriched human CD34⁺ cells was inhibited by pretreatment with anti-CXCR4 antibodies. Moreover, primitive CD34⁺CD38^−/lowCXCR4⁺cells also homed in response to a gradient of human stromal cell-derived factor 1 (SDF-1), directly injected into the bone marrow or spleen of nonirradiated NOD/SCID mice. Homing was also inhibited by pretreatment of CD34⁺ cells with antibodies for the major integrins VLA-4, VLA-5, and LFA-1. Pertussis toxin, an inhibitor of signals mediated by Gα_iproteins, inhibited SDF-1–mediated in vitro transwell migration but not adhesion or in vivo homing of CD34⁺ cells. Homing of human CD34⁺ cells was also blocked by chelerythrine chloride, a broad-range protein kinase C inhibitor. This study reveals rapid and efficient homing to the murine bone marrow by primitive human CD34⁺CD38^−/lowCXCR4⁺cells that is integrin mediated and depends on activation of the protein kinase C signal transduction pathway by SDF-1.