Previous reports debated the effects of differentiation on adenoviral vector (AdV) transduction efficiency and Cox-sackie-adenovirus receptor (CAR) expression. This prompted us to investigate the efficiency of AdV transduc-tion and CAR expression in human mesenchymal stem cells (hMSCs) and their differentiated progeny. Current results revealed high efficiency (>90%) of AdV transduction and a consistent level of CAR expression in hMSCs by the use of AdV carrying the enhanced green fluorescent protein reporter gene. Competition of CAR with blocking monoclonal antibody RmcB resulted in a reduction in transduction efficiency, indicating the CAR involvement in transduction of hMSCs. The cells were then induced to differentiate into bone, fat, or neural cells, and results demonstrated that the differentiation was accompanied with a consistent decline in AdV transduction and a decrement in CAR expression. Cells were infected with AdV and then induced into differentiation, and results demonstrated that transduced cells preserved differentiation potentials and still had transgene expression in a subpopulation of cells for 4 weeks and even in tested lineage-specific differentiation. According to the present investigation, undifferentiated hMSCs can serve as a gene-delivering system, and gene transfer into hMSCs before differentiation can resolve the difficulties in transduction of their differentiated progeny.