NOD-scid mice have been widely used as recipients in the xenograft assay for human hematopoietic stem cells (HSCs). One major problem with the strain is the low level of engraftment except when large numbers of cells are injected. This is probably caused by the presence of residual natural killer (NK) cell cytotoxic activity. NOD-scid/beta 2 microglobulin (B2m)null mice have been reported to have reduced NK cell cytotoxic activity and support higher levels of human cell engraftment. However, use of this strain of mice is limited by their difficulty in breeding and short life span caused by early development of lymphomas, which is accelerated by irradiation. Another immune-incompetent mouse model, NOD-recombination activating gene (Rag1)null mice allow longer observation of human cell engraftment than NOD-scid mice and are easier to breed. Genetic crossing of perforin (Prf) structure gene-targeted mutation onto NOD-Rag1null strain results in absence of NK cell cytotoxic function. In these mice, NK cells are not capable of killing target cells because of the absence of Prf, the major mediator of cytotoxic activity. We have tested the use of NOD-Rag1nullPrf1null mice as recipients of long-term xenograft assay for human HSCs by adopting Yoder’s method of conditioning newborn mice with minor modifications. Pregnant NOD-Rag1nullPrf1null dams were treated with 22.5mg/kg busulfan in 20% dimethylsulfoxide in Hank’s BSS on day 17.5 and 18.5 pc via subcutaneous injection. On the day of delivery, mononuclear cells (MNCs) were isolated from human cord blood (n=3) by density gradient centrifugation and T cell-depleted MNCs were separated by using mouse anti-human CD3, CD4, and CD8 antibodies and sheep anti-mouse IgG immunomagnetic beads to prevent preferential T cell engraftment. The busulfan-exposed pups were transplanted with 4–5 million T cell-depleted MNCs via the facial vein. At 6 months post-transplantation, human cells were detected in the bone marrow of 4 out of 10 transplanted mice. The levels of human CD45+ cells in the bone marrow of engrafted mice were 79.9, 69.8, 60.5, and 7.4%, and those in the peripheral blood were 6.3, 5.8, 4.1, and 1.3%. Multilineage engraftment was confirmed by phenotypic analysis. Next, we tested the hypothesis that human cord blood HSCs have dye efflux activity by injecting T cell-depleted Rhodamine 123 (Rho)− or + cells into conditioned newborn NOD-Rag1nullPrf1null mice. Six-month engraftment was found only with the Rho− cells. Thus, conditioned newborn NOD-Rag1nullPrf1null mice provide an excellent model for assaying long-term engrafting human HSCs.