Abstract Fanconi anemia (FA) is characterized by bone marrow aplasia and myeloid leukemia. The identification of FA genes raises the potential of using gene transfer technology to introduce cDNAs into autologous HSCs. Current strategies using Moloney retroviruses require a 2–4 day ex vivo culture of HSC to facilitate stable integration of the transgene. However, ex vivo culture results in a time-dependent increase in apoptosis of Fancc−/− primitive HSC and mice reconstituted with the surviving cells have an increased risk of acquiring myeloid malignancies. Therefore we examined the potential of a recombinant foamy virus construct (MD9-FANCC-EGFP) to transduce murine Fancc −/− HSC in the absence of prestimulation. Forty-80% of progenitors that were in G0 – G1 at the time of transduction were transduced following a single 10–14 hr transduction. Aliquots of MD9-FANCC-EGFP transduced BM cells or cells encoding the EGFP transgene only were transplanted into irradiated recipient mice or recipients treated with IFN-g only. Four-six months following transplantation, recipient BM cells were isolated and clonogenic assays were established in a range of mitomycin c (MMC) concentrations. Fancc−/− progenitors encoding recombinant FANCC were found to have a similar resistance to MMC as wildtype (WT) controls while Fancc−/− progenitors encoding the reporter construct only retained a high sensitivity to MMC. To assess the potential of MD9-FANCC-EGFP to correct stem cell repopulating ability, we next utilized the competitive repopulating assay. The repopulating activity of MD9-FANCC-EGFP-transduced Fancc−/− stem cells was comparable to WT controls 18–24 months following transplantation in primary and secondary recipients. Additionally, while mice reconstituted with Fancc−/− cells transduced with the reporter construct had reduced repopulating ability as compared to the other groups, none of these recipients acquired myeloid malignancies. Collectively, these data provide in vivo evidence that an abbreviated transduction protocol utilizing a foamy-viral based vector allows efficient transduction of Fancc−/− HSC, and diminishes the selection pressure that occurs during ex vivo culture of Fancc−/− HSCs.