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Xenografting immunodeficient mice after low‐dose irradiation has been used as a surrogate human hematopoietic stem cell (HSC) assay; however, irradiation requires strict and meticulous animal support and can produce significant mortality rates, limiting the usefulness of this model. In this work, we examined the use of parenteral busulfan as an alternative conditioning agent. Busulfan led to dose‐dependent human HSC engraftment in NOD/LtSz‐ scid/IL2Rγ   null   mice, with marked improvement in survival rates. Terminally differentiated B and T lymphocytes made up most of the human CD45+ cells observed during the initial 5 weeks post‐transplant when unselected cord blood (CB) products were infused, suggesting derivation from existing mature elements rather than HSCs. Beyond 5 weeks, CD34+‐enriched products produced and sustained superior engraftment rates compared with unselected grafts (CB CD34+, 65.8% ± 5.35%, vs. whole CB, 4.27% ± 0.67%, at 24 weeks). CB CD34+ group achieved significantly higher levels of engraftment than mobilized CD34+‐enriched peripheral blood (PB CD34+). At 8 weeks, all leukocyte subsets were detected, yet human red blood cells (RBCs) were not observed. Transfused human red cells persisted in the chimeric mice for up to 3 days; an accompanying rise in total bilirubin suggested hemolysis as a contributing factor to their clearance. Recipient mouse‐derived human HSCs had the capacity to form erythroid colonies in vitro at various time points post‐transplant in the presence of human transferrin (Tf). When human Tf was administered singly or in combination with anti‐CD122 antibody and human cytokines, up to 0.1% human RBCs were detectable in the peripheral blood. This long evasive model should prove valuable for the study of human erythroid cells. S TEM  C ELLS   2009;27:175–182

Busulfan Produces Efficient Human Cell Engraftment in NOD/LtSz‐ Scid IL2Rγ Null Mice