Background: Retroviral vectors are commonly used for gene transfer applications and they represent an effective way to provide a sustained delivery of a bioactive factor in basic research and tissue engineering applications. Cells that have been transduced with retroviral vectors ex vivo are usually amplified on tissue culture plastic, for a prolonged period of time, in order to obtain sufficient cell numbers prior to the experiment of interest. However, the effect of the transgene product on the transduced cells, during this period of time, is rarely, if ever, investigated. The current study investigated if transduction with a VSG.G pseudotyped retroviral vector expressing human bone morphogenetic protein 2 (Rv.BMP-2) influences the gene expression profile of human bone marrow-derived mesenchymal stem cells (hMSCs) during monolayer proliferation. hMSCs that have been transduced with a VSG.G pseudotyped retroviral vector expressing enhanced green fluorescent protein (Rv.eGFP) served as controls. Results: It was confirmed that Rv.BMP-2 transduced hMSCs produce detectable amounts of bone morphogenetic protein 2 (BMP-2). Gene expression analysis revealed that the hypertrophic marker collagen X was down-regulated by approximately 50% and the chondrogenic marker Aggrecan was elevated almost 9-fold in Rv.BMP-2 transduced hMSCs if compared to Rv.eGFP transduced control cells. Interestingly, the same changes in gene expression were detected when hMSCs were exposed to 100 ng/ml of recombinant human BMP-2 and their gene expression profile was compared to control hMSC. Again, collagen X message was down-regulated and Aggrecan message was up-regulated. Conclusion: These results indicate that, when using integrating vectors and then expanding the cells after transduction, controls need to be carefully planned to ensure the results obtained during the 3D experiments are not due to artefacts created in response to the different cell proliferation conditions employed.