We examined the potential of ex vivo gene therapy to enhance bone repair using lentiviral vectors encoding either enhanced green fluorescent protein (EGFP) as a reporter gene or bone morphogenetic protein-2 (BMP-2) downstream of either the cytomegalovirus immediate early (CMV) promoter or the murine leukemia virus long terminal repeat (RhMLV) promoter derived from a murine retrovirus adapted to replicate in a rhesus macaque. In vitro, rat bone marrow stromal cells (BMSCs) transduced with Lenti-CMV-EGFP or Lenti-RhMLV-EGFP demonstrated over 90% transduction efficiency at 1 week and continued to demonstrate stable expression for 8 weeks. ELISA results demonstrated that lentivirus-mediated gene transfer into BMSCs induced stable BMP-2 production in vitro for 8 weeks. Increased EGFP and BMP-2 production was noted with the RhMLV promoter. In addition, we implanted BMSCs transduced with Lenti-RhMLV-BMP-2 into a muscle pouch in the hind limbs of severe combined immune deficient mice. Robust bone formation was noted in animals that received Lenti-RhMLV-BMP-2 cells at 3 weeks. These results demonstrate that lentiviral vectors expressing BMP-2 can induce long-term gene expression in vitro and new bone formation in vivo under the control of the RhMLV promoter. Prolonged gene expression may be advantageous when developing tissue engineering strategies to repair large bone defects.