The objective of the present work was to study the effect of interleukin-3 (IL-3) and Flt3 ligand (FL) over-expression in human cord blood CD34(+) cells on rescuing lethally irradiated mice by transplantation. CD34(+) cells were isolated from human umbilical cord blood (UCB) and infected with recombinant retrovirus expressing FL, IL-3 or FL/IL-3 genes, respectively. Stably transduced CD34(+) cells were transplanted i.v. into NOD/SCID/IL2rgamma(null) mice that had been conditioned with 8.0 Gy of irradiation. At 6 weeks post-irradiation, chimerism in the animal bone marrow (BM) and the spleen were investigated. Recovery of peripheral blood cell and animal survival were recorded 2, 4, and 6 weeks post-irradiation. The chimerism was further investigated by serial transplantation assay. At 6 weeks post-transplantation, each of the three transduced human UCB CD34(+) cell types could be observed in all examined BM of chimeric mice; however, more human cells could be found in BM, spleen and peripheral blood of those mice transplanted with CD34(+) cells over-expressing IL-3/FL cells. Over-expression of IL-3 and FL at mRNA and protein levels could be detected in the BM cells of chimeric mice. Accordingly, hIL-3/FL co-overexpressing mice showed greater recovery of peripheral blood counts as early as 2 weeks after irradiation, and a much higher survival rate (11/12) than other groups at 6 weeks post-irradiation. Serial transplantation assay indicated that human cord blood CD34(+) cells could recover potentials of proliferation and self-renewal after being transplanted into mice. Our results suggested that transplant of CD34(+) cord blood cells over-expressing IL-3/FL genes improved rescue of radiation-injured mice, which probably results from the synergistic effect between hIL-3 and hFL causing the rapid reconstitution of hematopoiesis.