Human CD133⁺ stem cells were injected into the bone marrow cavity of NOG (NOD Shi-SCID IL2Rγc^null) mice with or without preconditioning of busulfan in order to assess the efficiency of human CD133⁺ cells engraftment. Peripheral blood from CD133⁺-engrafted NOG mice was analyzed by flow cytometry. The results showed that human CD19⁺ B lymphocytes could be detected at 4 weeks post-transplantation, and human CD4⁺, CD8⁺ subsets of T lymphocytes, CD19^– CD14^– HLA-DR⁺ DCs and CD19^– CD14⁺ monocytes could be detected at 16 weeks post-transplantation. The survival rate of mice in busulfan-untreated group (100%) was slightly higher than that in the busulfan-pretreated group (83%) ( P > 0.05). However, the differentiation efficiency of CD133⁺ stem cells in busulfan-pretreated group was significantly higher than that in the untreated group ( P < 0.05). This data imply that CD133⁺ cells could be a good resource for a humanized mouse model, and the preconditioning of busulfan could be more conducive to accelerating the differentiation of human CD133⁺ cells in NOG mice by intra-bone marrow injection.