Human adipose tissue-derived stromal cells (ADSCs) are being evaluated for cardiovascular repair. We developed an ex vivo method for producing angiogenic ADSCs transduced with a self-inactivating lentiviral vector (LV) expressing the enhanced green fluorescence protein (EGFP) from an internal cytomegalovirus (CMV) promoter to track these cells after in vivo engraftment. ADSCs from visceral adipose tissue were transduced using a LV incorporating the Rous Sarcoma Virus (RSV) long terminal repeat (LTR) sequences and the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) to enhance EGFP gene expression. We compared infection protocols with non-concentrated lentiviral supernatant or pellet fractions after ultracentrifugation, testing transduction efficiency, and reporter gene expression by quantitative flow cytometry at 5 and 28 days. Transduction of ADSCs with pellet after ultracentrifugation provided the highest transduction rate [flow cytometry titers: 6.5 ± 0.3 × 105 transduction units (TU)/mL and 20 ± 1.2 × 106 TU/mL at day 5 with non-concentrated lentiviral supernatant and pellet, respectively, with titer in the supernatant after ultracentrifugation remaining undetectable]. Reporter gene expression did not affect cell viability, morphology, proliferation, differentiation, self-renewal, or angiogenic activity. Furthermore, reporter gene expression did not significantly affect Fas/CD95-induced apoptosis. The in vivo implantation of transduced ADSCs into a mouse ischemic leg model resulted in efficient engraftment and angiogenesis. ADSC gene labeling using LVs is feasible and efficient, without impairment of stem cell characteristics, cell engraftment, and angiogenic activity. Such transduced ADSCs can be efficiently tracked in vitro and in vivo and may serve as vehicle for therapeutic genes. © 2012 Springer Science+Business Media, LLC.