Little is known about the cell types or mechanisms that underlie the engraftment process. Here, we have examined parameters affecting the engraftment of purified human Lin-CD34+CD38- normal and AML cells transplanted at limiting doses into NOD/SCID recipients. Mice transplanted with 500 to 1000 Lin-CD34+CD38- cord blood (CB) or AML cells required the co-transplantation of accessory cells (ACs) or short-term in vivo cytokine treatment for engraftment, whereas transplantation of higher doses (>5000 Lin-CD34+CD38- cells) did not show these requirements suggesting that ACs are effective for both normal and leukemic stem cell engraftment in this model. Mature Lin+CD34- and primitive Lin-CD34+CD38+ cells were capable of acting as ACs even though no repopulating cells are present. Cytokine treatment of NOD/SCID mice could partially replace the requirement for co-transplantation of AC. Furthermore, no difference was seen between the percentage of engrafted mice treated with cytokines for only the first 10 days after transplant compared to those receiving cytokines for the entire time of repopulation. Surprisingly, no engraftment was detected in mice when cytokine treatment was delayed until 10 days posttransplant. Together, these studies suggest that the engraftment process requires pluripotent stem cells plus accessory cells or cytokine treatment which act early after transplantation. The NOD/SCID xenotransplant system provides the means to further clarify the processes underlying human stem cell engraftment.