Abstract Optimization of therapy for childhood acute lymphoblastic leukemia (ALL) requires a greater understanding of the cells that proliferate to maintain this malignancy because a significant number of cases relapse, resulting from failure to eradicate the disease. Putative ALL stem cells may be resistant to therapy and subsequent relapses may arise from these cells. We investigated expression of CD133, CD19, and CD38 in pediatric B-ALL. Cytogenetic and molecular analyses demonstrated that karyotypically aberrant cells were present in both CD133+/CD19+ and CD133+/CD19− subfractions, as were most of the antigen receptor gene rearrangements. However, ALL cells capable of long-term proliferation in vitro and in vivo were derived from the CD133+/CD19− subfraction. Moreover, these CD133+/CD19− cells could self-renew to engraft serial nonobese diabetic–severe combined immunodeficient recipients and differentiate in vivo to produce leukemias with similar immunophenotypes and karyotypes to the diagnostic samples. Furthermore, these CD133+/CD19− ALL cells were more resistant to treatment with dexamethasone and vincristine, key components in childhood ALL therapy, than the bulk leukemia population. Similar results were obtained using cells sorted for CD133 and CD38, with only the CD133+/CD38− subfraction demonstrating xenograft repopulating capacity. These data suggest that leukemia-initiating cells in childhood B-ALL have a primitive CD133+/CD19− and CD38− phenotype.