Oncoretroviral vectors require division of target cells for successful transduction. In the case of hematopoietic repopulating cells this can be achieved by cytokine stimulation using growth factor combinations which facilitate gene transfer and maintain engraftment. Interleukin-3 (IL-3) has been widely used in growth factor combinations, although more recent data in the mouse showed reduced engraftment in the presence of IL-3. Here, we used a competitive repopulation assay to study the influence of IL-3 and the early acting cytokines megakaryocyte growth and development factor (MGDF) and Flt3-ligand (Flt3-L) on gene transfer efficiency during ex vivo transduction of hematopoietic repopulating cells. In a direct comparison, baboon CD34-enriched cells were transduced on CH-296 fibronectin fragment in the presence of either IL-6, stem cell factor (SCF), Flt3-L, and MGDF or IL-3, IL-6, and SCF. Animals were followed for up to 55 weeks, and analysis of peripheral blood leukocytes by semiquantitative polymerase chain reaction showed that both cytokine combinations achieved marking of repopulating cells. A trend toward increased gene marking, especially early after transplant (P = 0.06), was seen with the combination of IL-6, SCF, Flt3-L, and MGDF. However, the highest gene marking was achieved when IL-3 was combined with early acting cytokines, suggesting that the difference observed in this study was probably due to the addition of MGDF and Flt3-L and not due to a negative effect of IL-3 on engraftment.