Abstract Ex vivo expansion of cord blood [CB] hematopoietic stem cells [HSC] may increase the number of patients eligible for allogeneic transplants, by making CB a reliable source of HSC for transplantation. We previously showed that CB-HSC can be significantly expanded with flt-3 ligand [FL] and c-mpl ligand [ML]; 106 input CD34+ cells increase to 108 CD34+ cells by 8-9 weeks of culture. However, it is essential to know the function of expanded CB HSC cells prior to clinical usage. To assess the in vivo function of expanded CB HSC, cells from expansion cultures were injected into irradiated [375 cGy] non-obese diabetic/severe combined immune deficient [NOD-scid] mice. Uncultured CD34+ UCB served as engraftment controls. Engraftment was analyzed 5-8 weeks post-injection by two-color staining of bone marrow cells with antibodies to human CD45 [FITC] and selected CD antigens [PE]. Cells grown from 2 to 16 weeks engraft NOD-scid mice, as evidenced by human CD45+ cells in the marrow, giving rise to mature human leukocytes. Surprisingly, cells from 2-week cultures generate few CD34+ cells in the marrow of NOD-scid mice, while mice engrafted with cells from cultures ≥3 weeks appear similar to uncultured UCB controls. Kinetic analysis of CD38 expression on ex vivo expanded CB-HSC using 3-color flow cytometry shows that the CD34+ cells from uncultured UCB are predominantly CD38lo, but there is a shift to CD34+/CD38+ cells during the first 2 weeks of culture. At later time points, the CD38+ population diminishes, so that virtually all CD34+ cells are CD38lo. Since CD34+/CD38+ stem cells are considered more mature than CD34+/CD38lo HSC, our results correlated CD38 expression to CD34+ engraftment potential. These findings indicate that short-term cultures (10-14 days) lead to a decline in CB HSC activity and impaired engraftment, but extended culture conditions lead to a return of CD34+/CD38lo stem cells, and restoration of normal engraftment potential by these cultured CB HSC.