Mesenchymal stem cells (MSCs) represent a source of pluripotent cells that are already in various phases of clinical application. However, the use of MSCs in tissue engineering has been hampered largely due to their limitations, including low proliferation, finite life span, and gradual loss of their stem cell properties during ex vivo expansion. Nanog and Oct4 are key transcription factors essential to the pluripotent and self-renewing phenotypes of undifferentiated embryonic stem cells (ESCs). To determine whether Nanog and Oct4 improve human bone marrow-MSC quality, we therefore established stable Nanog and Oct4 overexpressing MSCs using a lentiviral system and showed that this promoted cell proliferation and enhanced colony formation of MSCs. In differentiating MSCs, Nanog, and Oct4, overexpression had converse effects on adipogenesis of MSCs and Nanog overexpression slowed down adipogenesis, whereas Oct4 overexpression improved adipogenesis. Nanog and Oct4 overexpression both improved chondrogenesis. Microarray data showed many differences in transcriptional targets in undifferentiated MSCs overexpressing Nanog and Oct4. These results provide insight into the improvement of the stemness of MSCs by genetic modification with stemness-related genes.