OBJECTIVE The potential of human mesenchymal stem cell-like stroma prepared from placental/umbilical cord blood for hematopoietic regeneration by X-irradiated hematopoietic stem cells is herein assessed. METHODS Placental/umbilical cord blood-derived mesenchymal stem cell-like stromal cells were applied to a regenerative ex vivo expansion of X-irradiated human CD34+ cells in a serum-free liquid culture supplemented with a combination of interleukine-3 plus stem cell factor plus thrombopoietin. RESULTS The total number of cells and of lineage-committed myeloid hematopoietic progenitor cells generated in the co-culture of both non-irradiated and X-irradiated cells with stromal cells was significantly higher than those in the stroma-free culture. In addition, the number of CD34+ cells and CD34+/CD38− cells, immature hematopoietic stem/progenitor cells also increased more than the stroma-free culture. The stromal cells produced various types of cytokines, although there was little difference between the co-cultures of non-irradiated and X-irradiated cells with stromal cells. Furthermore, when X-irradiated cells came in contact with stromal cells for 16 h before cytokine stimulation, a similar degree of hematopoiesis was observed, thus suggesting the critical role of cell-to-cell interaction. CONCLUSIONS The present results showed the potential efficacy of human mesenchymal stem cell-like stroma for hematopoietic regeneration from irradiated hematopoietic stem/progenitor cells.