Bone marrow derived-stromal cells (BMSCs) are pluriplotent progenitors for a variety of cell types, including osteoblast, chondrocyte, adipocyte, and so on. Stem cell research has enormous potential in the future clinical treatment of a wide range of diseases. Tracking stem cell localization, survival, differentiation, and proliferation after transplantation in living subjects is essential for understanding stem cell biology and physiology. However, we don’t have exact evaluation methods and safeguards for clinical application. In this study, we investigated tracking BMSCs differentiation, localization, toxicity, and migration in vivo. The fluorescent vector used in our studies did not affect BMSCs viability or their ability to undergo osteogenic and adipogenic differentiation in vitro. During differentiation, EGFP-BMSCs by Oil Red O and Alizarin Red S were stained. EGFP-BMSCs were transplanted into the femoral region in autologous rabbit. After one month, these cells were detectable by confocal microscopy and RTPCR. Transplanted EGFP-BMSCs were not detected another organs (spleen, kidney, liver, and muscle) in immunohistochemistry and RT-PCR. In organ-function test and cell-toxicity examination, there is no difference between the before and after EGFP-BMSCs transplantation. we observed that transplanted EGFP-BMSCs were not affected cell-toxicity and migration. This results offer some evaluation methods and safeguards for clinical application using BMSCs. In further study, it will be needed to test reproductive and developmental toxicity after transplantation and observe migration of EGFP-BMSCs after transplantation.