To investigate the clinically applicable conditions that support substantial expansion of both primitive and more mature hematopoietic cells of umbilical cord blood (UCB) for transplantation in adults, enriched CD34+ cells from 8 fresh UCB samples and 4 expanded UCB products were cultured in defined serum-free medium (QBSF-60) in the presence of a cytokine combination of SCF, Flt-3-ligand (FL), thrombopoietin (TPO), IL-3 for up to 2 weeks. Fresh medium with cytokines was supplemented or exchanged at day 4, day 7, and day 10. The proliferative response was assessed at day 7, day 10, and day 14 by evaluating the following parameters: nucleated cell (NC), clonogenic progenitors (colony-forming unit-granulocyte-macrophage [CFU-GM], burst-forming unit-erythrocyte [BFU-E], CFU-GEMM, and high-proliferative potential colony-forming cell [HPP-CFC]), immunophenotypes (CD34+ cells and CD34+ subpopulations), and LTCIC. Simultaneously numerical expansion of various stem/progenitor cells, including primitive CD34+ CD38- HLA-DR- subpopulation and LTCIC, CD34+ cells, and clonogenic progenitors to mature nucleated cells, were continuously observed during the culture. An average 103.32±71.37×106 CD34+ cells (range 10.12×106-317.9×106) could be obtained from initial 1.72±1.13×106 UCB CD34+ cells after 10-14 days cultured under the described conditions. Sufficient CD341 cells (＞50.0×106) for transplantation in adults would be available in all but one UCB collections after 10-14 days expansion. The expanded CD34+ cells sustained most of the in vitro characteristics of initial unmanipulated CD34+ cells, including clonogenic efficiency (of both primitive and committed progenitors), the proportion of CD34+ CD38-HLA-DR- subpopulation, and the expansion potential. Initial addition of IL-3 to the cocktail of SCF+ FL+ TPO had positive effects on the expansion of both primitive and, especially, the more mature hematopoietic cells. It accelerated the expansion speed and shortened the optimal culture time from 14 days to 10 days. These results indicated that our proposed shortterm culture system, consisting of QBSF-60 serum-free medium with a simple early acting cytokine combination of SCF+ FL+TPO, could substantially support simultaneous expansion of various stem/progenitor cell populations involved in the different phases of engraftment. It would be a clinically applicable protocol for ex vivo expansion of CD34+ UCB cells.