Ex vivo expansion of hematopoietic stem cells would be useful for bone marrow transplantation and gene therapy applications. Toward this goal, we have investigated whether retrovirally-transduced murine stem cells could be expanded in culture with hematopoietic cytokines. Bone marrow cells were transduced with retroviral vectors expressing either the human multidrug resistance 1 gene (HaMDR1), a variant of human dihydrofolate reductase (HaDHFR), or both MDR1 and DHFR in an internal ribosomal entry site (IRES)-containing bicistronic vector (HaMID). Cells were then expanded for 15 days in cultures stimulated with interleukin (IL)-3, IL-6, and stem cell factor. When very low marrow volumes were injected into lethally irradiated recipient mice, long-term reconstitution with 100% donor cells was seen in all mice injected with HaMDR1- or HaMID-transduced cells. By contrast, engraftment with HaDHFR- or mock-transduced cells ranged from partial to undetectable despite injection of significantly larger marrow volumes. In addition, mice transplanted with expanded HaMDR1- or HaMID-transduced stem cells developed a myeloproliferative disorder that was characterized by an increase in abnormal peripheral blood leukocytes. These results show that MDR1-transduced stem cells can be expanded in vitro with hematopoietic cytokines, but indicate that an increased stem cell division frequency can lead to stem cell damage.