Human umbilical cord blood (CB) has been used successfully in stem cell transplantation. A subpopulation of CD34+ cells expresses chemokine receptor CXCR4 which is critical for bone marrow engraftment in human hematopoietic stem cells. Here, we demonstrate the effect of short-term culture on CXCR4 expression on umbilical CB-derived CD34+ cells and subsequent engraftment capability in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Surface CXCR4 expression on CD34+ cells increased after incubating the cells in medium alone for 2 h; this effect was blocked by the addition of AMD3100. No difference in CXCR4 mRNA expression was noted after incubating CD34+ cells in culture for 2 h, although these cells showed significantly increased transmigrational activity toward SDF-1 and homing activity in NOD/SCID mice. Furthermore, cultured human CD34+ cells showed improved engraftment into the bone marrow of NOD/SCID mice compared to noncultured or AMD3100-treated CD34+ cells. These observations suggest that increased cell surface expression of CXCR4 on CD34+ cells improved the engraftment of human umbilical CB cells into bone marrow through enhanced homing activity.