In this study, we developed a methodology to improve the survival, vascular differentiation and regenerative potential of umbilical cord blood (UCB)-derived hematopoietic stem cells (CD34⁺ cells), by co-culturing the stem cells in a 3D fibrin gel with CD34⁺-derived endothelial cells (ECs). ECs differentiated from CD34⁺ cells appear to have superior angiogenic properties to fully differentiated ECs, such as human umbilical vein endothelial cells (HUVECs). Our results indicate that the pro-survival effect of CD34⁺-derived ECs on CD34⁺ cells is mediated, at least in part, by bioactive factors released from ECs. This effect likely involves the secretion of novel cytokines, including interleukin-17 (IL-17) and interleukin-10 (IL-10), and the activation of the ERK 1/2 pathway in CD34⁺ cells. We also show that the endothelial differentiation of CD34⁺ cells in co-culture with CD34⁺-derived ECs is mediated by a combination of soluble and insoluble factors. The regenerative potential of this co-culture system was demonstrated in a chronic wound diabetic animal model. The co-transplantation of CD34⁺ cells with CD34⁺-derived ECs improved the wound healing relatively to controls, by decreasing the inflammatory reaction and increasing the neovascularization of the wound.