Primitive human hematopoietic cells in granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood (MPB) are more difficult to transduce compared to cells from umbilical cord blood. Based on the hypothesis that MPB cells may require different stimulation for efficient retroviral infection, we compared several culture conditions known to induce cycling of primitive hematopoietic cells. MPB-derived CD34(+) cells were stimulated in the presence or absence of the murine fetal liver cell line AFT024 in trans-wells with G-CSF, stem cell factor (SCF), and thrombopoietin (TPO) (G/S/T; 100 ng/ml) or Flt3-L, SCF, interleukin (IL)-7, and TPO (F/S/7/T; 10-20 ng/ml), and transduced using a GaLV-pseudotyped retroviral vector expressing the enhanced green fluorescence protein (eGFP). Compared to cultures without stroma, the presence of AFT024 increased the number of transduced colony-forming cells (CFC) by 3.5-fold (with G/S/T), long-term culture-initiating cells (LTC-IC) by 4.6-fold (with F/S/7/T), and nonobese diabetic/severe immunodeficiency disease (NOD/SCID)-repopulating cells (SRC) by 6.8-fold (with F/S/7/T). Similar numbers of long-term culture-initiating cells (LTC-IC) and SRC could be transduced using AFT024-conditioned medium (AFT-CM) or a defined medium that had been supplemented with factors identified in AFT-CM. Finally, using our best condition based on transduction with the gibbon ape leukemia virus (GaLV)-pseudotyped vector, we demonstrate a 33-fold higher level of gene transfer (p < 0.001) in SRC using an RD114-pseudotyped vector. In summary, using an optimized protocol with low doses of cytokines, and transduction with an RD114 compared to a GaLV-pseudotyped retroviral vector, the overall number of transduced cells in NOD/SCID mice could be improved 144-fold, with a gene-transfer efficiency in SRC of 16.3% (13.3-19.9; n = 6).