In umbilical cord blood (UCB) transplantation, the number of nucleated cells per kilogram is a major predictive and critical factor of hematopoietic recovery. Thus, ex vivo expansion of hematopoietic UCB progenitors could potentially accelerate engraftment. Whereas Flt-3 ligand (FL), stem cell factor (SCF), and thrombopoietin (TPO) are considered indispensable, the role of interleukin 3 (IL-3) is still controversial: it has been reported either to support or abrogate the reconstituting ability of stem cells. By adding IL-3 we aimed to enhance the amplification of early and committed progenitor cells without impairing the long-term engraftment of stem cells. Demonstrating a positive impact of IL-3 on the proliferation of all progenitor subsets, the amplification of CD34+ UCB cells was increased 20.9-fold +/- 5.4 (mean +/- standard error) in serum-free culture with FL, SCF, TPO, and IL-3 as opposed to 9.3-fold +/- 3.2 without IL-3 after 7 days. If IL-3 was included, primitive long-term culture-initiating cells and committed colony-forming cells were expanded 16.3-fold +/- 5.5 and 18.1-fold +/- 2.4, respectively, compared to 12.6-fold +/- 5.6 and 9.1-fold +/- 2.0 without IL-3. Analysis of cultured CD34+ UCB cells in sublethally irradiated nonobese diabetic/severe combined immunodeficient mice confirmed that cultured cells had preserved their repopulating potential. After 6 weeks, all mice showed multilineage engraftment with their bone marrow containing an average of 45% human CD45+ cells of the unmanipulated sample, 43% of cells after culture in the presence of IL-3, and 27% of cells after culture without IL-3. In combination with early acting cytokines, IL-3 therefore improves the ex vivo expansion of UCB stem and progenitor cells without impairing their engraftment potential.