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The development of ex vivo expansion of hematopoietic stem cells (HSCs) is a promising approach to restore the required bone marrow function of patients with hematological disorders. Previously, we have reported the development of an optimized serum-free and cytokines-limited defined medium using statistic methodology for umbilical cord blood-derived HSC expansion. The aim of this study was to analyze further the characteristics and functions of cells in vitro and in vivo when cultured in this defined medium. After a 7-day batch culture, the average absolute fold expansions for CD133⁺ cells, CD34⁺CD133⁺ cells, CD34⁺CD38^– cells, CD133⁺CD38^– cells, CD34⁺CXCR4⁺ cells, CD133⁺CXCR4⁺ cells, and long-term culture-initiating cells were 21-, 20-, 723-, 618-, 160-, 384-, and 8-fold, respectively. The high enrichment of CD38^– cells and CXCR4⁺ cells of the CD34⁺ subpopulation provided a very early uncommitted HSC proliferation and homing ability. Furthermore, the expanded cells showed a high level of telomerase activity to maintain their telomere length and repopulated the lethally irradiated NOD/SCID mice in vivo. These results indicated that the cytokineslimited expanded cells from CD133⁺ cells could substantially support simultaneous expansion of various stem/progenitor cells and engraft with the expanded cells from a low number of HSCs initially.