We have previously identified a novel circulating embryonic blood stem cell that is distinct from both adult bone marrow (BM) and cord blood (CB) derived human repopulating cells, that may serve as an ideal target cell for in utero stem cell therapy. Towards this goal, we examined the ability to maintain these fetal blood (FB) stem cells in ex vivo culture conditions previously optimized for repopulating cells derived from later stages of human hematopoietic ontogeny. FB cells were evaluated for Scid-Repopulating capacity (SRC) before and after 4 days of serum free ex vivo culture, using control growth factor (OF) combinations of SCF, FLT-3L, IL-3, IL-6 and G-CSF (GF1). In contrast to CB-SRC, circulating FB repopulating cells could not be maintained under these conditions. FB-SRC were further compared for maintainance of repopulating function in ex vivo conditions using GF1 combinations, GFl+Oncostatin M, or with SCF+FLT-3L alone. Although the number and type of FB hematopoietic progenitors were similar in all conditions, ex vivo culture with SCF+FLT-3L resulted in reduced proliferation. Despite this reduction in cell expansion, the combination of SCF+FLT-3L was able to sustain FBSRC. These results demonstrate SCF and FLT-3L are sufficient in retaining FB stem cell capacity, and that additional cytokines required for adult and CB derived stem cell maintainance induce loss of these circulating stem cells. Our study provides novel insights into the requirements of early fetal stem cells that are essential in creating ex vivo conditions nessessary for future in utero stem cell gene therapy, and illustrates the biological distinctiveness of FB stem cells as compared to other stages of human ontogeny.