Acute lymphoblastic leukemia (ALL) is a heterogeneous disorder, and primary drug resistance and relapse are thought to be the main causes for treatment failure in ALL patients. For these refractory or relapsed patients, there is an increasing demand to identify novel therapeutic approaches, which will highly rely on the use of xenotransplantation models in translational research. Given the critical role that the spleen plays in the hematopoiesis and lymphopoiesis in adult mice, intrasplenic inoculation of ALL cells into immunodeficient mice may represent a feasible route for leukemic xenotransplantation. In the present study, engraftments via intrasplenic inoculation in anti-mCD122 mAb conditioned NOD/SCID mice were achieved in 5 out of 11 cases, and the engrafted cells reconstituted a complete leukemic phenotype. The engrafted cells sustained the self-renewal capacity of leukemia-initiating cells as tested by serial xenotransplantation and can be used for evaluation of antileukemic drugs. These data suggest that the combination of intrasplenic inoculation and the targeted depletion of CD122(+) cells could provide a novel approach for the xenotransplantation of ALL cells in NOD/SCID mice. Furthermore, this model can be used for stem cell research, long-term analysis of engraftment kinetics and in vivo drug tests.