HIV-based lentiviral vectors (LV) have become powerful tools for in vivo gene transfer and gene expression in non-dividing cells after local injection. However, the potential of in vivo bone marrow stem cell gene transfer by “in situ” bone cavity injection has not been well unexplored. This in vivo approach could take full advantage of any source of stem cells present in bone cavity and avoid many of the difficulties encountered by ex vivo hematopoietic stem cell (HSC) gene transfer. To determine if intra bone marrow (iBM) injection of LV can efficiently transduce BM stem cells, a 3rd-generation self-inactivating LV, containing eGFP regulated transcriptionally by a CMV promoter, was used to inject intra-femorally into adult Boy/J Ly45.1 mice (at 2 x106 IU/mouse). Blood counts were normal in all LV-injected mice (n=4) and buffer-injected mice (n=4). GFP-expressing peripheral blood leukocytes (PBL) were observed in both myeloid (up to 4%) and lymphoid subpopulations (up to 2%) 3-month post injection. To evaluate gene transfer and transgene expression in multi-potential progenitors, the colony-forming cell (CFC) assay was performed using low-density bone marrow 4-months post injection, resulting in 4–5% GFP+ CFU-granulocyte macrophage (CFU-GM) and CFU-granulocyte, erythroid, macrophage and megakaryocyte (CFU-GEMM). This was consistent with the 4-color FACS analysis data demonstrating that up to 3.7% of HSC/progenitors (Lin−c-kit+Sca1+ cells) from LV-injected mice expressed GFP transgene 4-months post injection. To further evaluate HSC gene transfer, BM from injected mice was transplanted into lethally irradiated adult C57/Bl6 (Ly45.2) mice. Four months later, successful engraftment was demonstrated in all BM recipients with 97–99% donor-derived cells in PBL and 91–93.3% in BM. Moreover, significant levels of GFP+ CFU were observed in all recipients ranging from 8.4 ± 2.3% to 17.7 ± 4.2%. To further study the LV-mediated gene transfer profile in HSC, mesenchymal progenitors and systemic distribution by intra-femoral injection, molecular analyses are in progress including real-time QPCR and clonality analysis by LAM-PCR. Our results indicate successful LV-mediated gene transfer into HSC by iBM injection. These data would potentially open a door to a novel approach for treatment of human diseases, but also provide a new tool to study adult stem cell plasticity and the nature of hematopoiesis.