Cotransplantation of ex vivo-expanded mesenchymal stem cells (MSC) together with hematopoietic stem cells hastens hematopoietic recovery in animal models and potentially humans. The in vivo mechanisms of migration, homing and long-term viability of MSC following implantation are not well understood. Reporter gene labeling would permit the tracking of transplanted MSC in vivo through noninvasive imaging techniques, namely fluorescence, bioluminescence, and nuclear imaging. Accordingly, a gibbon ape leukemia virus (GALV)-pseudotyped retrovirus was utilized to transfer a triple fusion gene reporter system, expressing enhanced green fluorescent protein (eGFP), luciferase, and herpes simplex virus type one thymidine kinase (HSV1-tk) into MSC. Human MSC were isolated and propagated from normal bone marrow aspirates by culturing ficoll-separated fractions in alpha-MEM medium supplemented with 20% fetal bovine serum. FACS analysis confirmed the MSC phenotype, with cells expressing HLA-Class I, CD105, CD73, CD90 and CD166 antigens and not expressing HLA-Class II, CD80, CD31, CD34 and CD45 antigens. No significant phenotypic changes were observed in early and late passage numbers of MSC. MSC were transduced during their exponential growth phase (as determined by growth curve measurements), with virus-containing medium (VCM) from the PG13 packaging cells (targeting Pit-1 receptors) for 24 hours in the presence of different concentrations of the cationic polymer, polybrene, ranging from 2 μg/ml to 12μg/ml. The 2μg/ml concentration appeared to be optimal for transduction of MSC. VCM was harvested following a 24-hour incubation of the PG13 packaging cells at 32°C or 37°C. These initial experiments suggested that incubation at 32°C appeared to be optimal for virus production. Repeated VCM exposure at 24-hour intervals for 2 or 3 days did not increase the percentage of transduced cells as determined by flow cytometry (eGFP-positive). VCM harvested at 32°C added to MSC in the presence of 2μg/ml polybrene, yielded a 62% transduction efficiency following one round of exposure. U87 human glioma cells were used throughout as a positive control to verify retrovirus efficacy and demonstrated >90% transduction efficiency. MSC clones highly expressing triple reporter genes are being selected and propagated in our laboratory to image transduced cells in NOD/SCID mice, thereby providing a better understanding of the potential role of these cells in transplantation processes.