Umbilical cord blood (UCB) is a readily available source of hematopoietic stem cells for transplantation. UCB hematopoietic SCT for average- and large-sized patients is often limited by the number of cells available in a single unit. To address this limitation, we performed experiments to determine if adjunctive therapy with third-party human allogeneic cells enhances the engraftment of human UCB in immunodeficient mice. UCB cells with or without sequential infusion of irradiated third-party allogeneic cells were used in transplantation studies of NOD/SCID and NOD/SCID-IL2Rγ null mice. We studied the impact of irradiated allogeneic cells on colony formation in vitro using long-term culture assays also. Our studies demonstrate that short- and long-term UCB engraftment of immunodeficient mice is enhanced by irradiated allogeneic cells. Secondary transplants demonstrate the durability of engraftment. These preclinical studies support the further development of irradiated allogeneic cells as an adjunct to single UCB transplantation when limiting numbers of cells are available.