Strategies to enhance the expansion of umbilical cord blood hematopoietic stem and progenitor cells (HSPCs) are crucial to enable their widespread application to adults and to overcome important limitations, such as delayed engraftment. Osteoblasts regulate HSPCs under steady-state and also under stress conditions, when HSPCs undergo numerous cycles of expansion. We hypothesized that osteoblasts could provide better stimulation for the expansion of multipotent HSPCs and subsequent hematopoietic recovery than mesenchymal stromal cells. Hence, we assessed the growth and engraftment modulatory activities of mesenchymal stromal cell-derived osteoblasts (M-OSTs) on hematopoietic progenitors. Mesenchymal stromal cells and M-OSTs favored the maintenance of CD34(+) cells. The expansion of cord blood CD34(+) cells and myeloid progenitors was highest in cultures supplemented with unfiltered M-OST-conditioned medium (M-OST CM). In addition, increased expression of cell surface receptors important for the homing of progenitors to the bone marrow, C-X-C chemokine receptor type 4 and lymphocyte function-associated antigen 1, was observed in CM-based cultures. Additionally, M-OST CM positively modulated the engraftment properties of expanded progenitors. Most notably, although human platelet levels remained steady in the first 2 weeks in mice transplanted with HSPCs expanded in standard medium, levels in mice transplanted with M-OST CM HSPCs rose continuously. Consistent with this, short-term human progenitor reconstitution was consistently greater in M-OST recipients. Finally, cytokine array-based profiling revealed increases in insulin-like growth factor binding protein 2, chemokines, and myeloid stimulating cytokines in M-OST CM. In conclusion, this study suggests that M-OSTs represent a new underappreciated source of feeder cells for the expansion of HSPCs with enhanced thrombopoietic activity. (C) 2014 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc.