We examined cell culture conditions with various combinations of cytokines including thrombopoietin (TPO) to obtain the most efficient transduction of recombinant retrovirus vectors into G-CSF-mobilized blood CD34+ cells which were obtained from children and purified with an Isolex 50 system (Baxter; Deerfield, IL). Three different 4-day culture conditions for the stimulation of CD34+ cells were compared in terms of a cell-cycle analysis by fluorometry and gene transduction efficiency as determined by resistance to G418 and NeoR polymerase chain reaction (PCR) for individual colony-forming unit-granulocyte/macrophage (CFU-GM) grown in a methylcellulose culture system. The cytokines tested were: A) interleukin (IL)-6 + stem cell factor (SCF); B) IL-3 + IL-6 + SCF, and C) IL-3 + IL-6 + SCF + TPO. Without a cell culture, the percentage of CD34+ cells in the cell cycle (the percentage of cells in phases S and G2/M) was 4.6%. After a four-day culture (n = 5), this value increased with the addition of IL-3 (22%) or IL-3 + TPO (27%, p < 0.05) as compared to that with the baseline cocktail of IL-6 + SCF (15%). The cell number uniformly increased approximately 10-fold in each culture condition. The average efficiency of gene transfer into incubated CD34+ cells with the corresponding combinations of cytokines was, respectively, 57%, 47%, and 30% for G418-screened CFU-GM and 72%, 68%, and 51% for polymerase chain reaction-positive CFU-GM. A statistically significant difference (p < 0.01) was found for G418/CFU-GM with IL-3 + IL-6 + SCF (57%) versus IL-3 + IL-6 + SCF + TPO (30%). Hence, it is likely that the increased cell proliferation produced by the addition of TPO was not necessarily translated into an increased rate of retroviral-mediated gene transduction, possibly because TPO preferentially induced the differentiation of stem cells into mature progenitors in these culture systems.