Homing and repopulation of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice by enriched human CD34⁺stem cells from cord blood, bone marrow, or mobilized peripheral blood are dependent on stromal cell-derived factor 1 (SDF-1)/CXCR4 interactions. Recently, human cord and fetal blood CD34⁺CD38⁻CXCR4⁻ and CXCR4⁺ cells, sorted with neutralizing anti-CXCR4 monoclonal antibody (mAb), were shown to have similar NOD/SCID repopulation potential. Herein we report that human cord blood CD34⁺CXCR4⁺ (R4⁺) and CD34⁺CXCR4⁻ (R4⁻) subsets, sorted with neutralizing anti-CXCR4 mAb, engrafted NOD/SCID mice with significantly lower levels of human cells compared with nonsorted and SDF-1–migrated CD34⁺ cells. Coinjection of purified cells with 10 μg anti-CXCR4 mAb significantly reduced engraftment of all CD34⁺ subsets, and 50 μg completely abrogated engraftment by R4⁻ and CD34⁺ cells. Importantly, R4⁻ cells harbor intracellular CXCR4, which can be rapidly induced to cell surface expression within a few hours. Moreover, 48 hours of cytokine stimulation resulted in up-regulation of both cell surface and intracellular CXCR4, restoring migration capacities toward a gradient of SDF-1 and high-level NOD/SCID repopulation potential. In addition, homing of sorted R4⁻ cells into the murine bone marrow and spleen was significantly slower and reduced compared to CD34⁺ cells but yet CXCR4 dependent. In conclusion, R4⁻ cells express intracellular CXCR4, which can be functionally expressed on the cell membrane to mediate SDF-1–dependent homing and repopulation. Our results suggest dynamic CXCR4 expression on CD34⁺ stem and progenitor cells, regulating their motility and repopulation capacities.