Flt3 Ligand (FL) and IL-6 are multifunctional cytokines implicated in normal hematopoiesis and ex vivo expansion of hematopoietic stem cells. Retroviral vectors are useful for stable expression of genes in many cells. Here, we aimed to produce retroviral vectors directing expression of human FL and IL-6 genes. Recombinant retroviral vectors containing human genes for FL and IL-6 were constructed using a retroviral vector pLXSN. Recombinant retroviruses were produced from GP2-293 cells with the aid of pseudo-envelope protein gene VSV-G, and efficiently transduced to a mouse stromal cell line OP9. Genetically modified OP9 cells clearly showed expression of human FL or IL-6 gene at the mRNA level determined by RT-PCR. Based on the results from ELISA, human FL and IL-6 were detected in the cell culture medium of OP9/FL and OP9/IL-6 cells, respectively. As the recombinant human FL and IL-6 proteins are successfully produced and secreted to the culture medium, this system can be useful in future application such as ex vivo expansion of hematopoietic stem cells and differentiation of embryonic stem cells.