Culture of mesenchymal stem cells (MSCs) under conditions promoting proliferation and differentiation, while supporting genomic and epigenetic stability, is essential for therapeutic use. We report here the extent of genome-wide DNA gains and losses and of DNA methylation instability on 170 cancer-related promoters in bone marrow (BM) MSCs during culture to late passage in medium containing fetal bovine serum (FBS) or autologous serum (AS). Comparative genomic hybridization indicates that expansion of BMMSCs elicits primarily telomeric deletions in a subpopulation of cells, the extent of which varies between donors. However, late passage cultures in AS consistently display normal DNA copy numbers. Combined bisulfite restriction analysis and bisulfite sequencing show that although DNA methylation states are overall stable in culture, AS exhibits stronger propensity than FBS to maintain unmethylated states. Comparison of DNA methylation in BMMSCs with freshly isolated and cultured adipose stem cells (ASCs) also reveals that most genes unmethylated in both BMMSCs and ASCs in early passage are also unmethylated in uncultured ASCs. We conclude that (i) BMMSCs expanded in AS or FBS may display localized genetic alterations, (ii) AS tends to generate more consistent genomic backgrounds and DNA methylation patterns, and (iii) the unmethylated state of uncultured MSCs is more likely to be maintained in culture than the methylated state.