Mesenchymal stem cells (MSCs) are of particular interest because they are being tested using cell and gene therapies for a number of human diseases. MSCs represent a rare population in tissues. Therefore, it is essential to grow MSCs in vitro before putting them into therapeutic use. This is compromised by senescence, limiting the proliferative capacity of MSCs. We analyzed the in vitro senescence of rat MSCs, because this animal is a widespread model for preclinical cell therapy studies. After initial expansion, MSCs showed an increased growth doubling time, lost telomerase activity, and expressed senescence-associated beta-galactosidase. Senescence was accompanied by downregulation of several genes involved in stem cell self-renewal. Of interest, several genes involved in DNA repair also showed a significant downregulation. Entry into senescence occurred with characteristic changes in Retinoblastoma (RB) expression patterns. Rb1 and p107 genes expression decreased during in vitro cultivation. In contrast, pRb2/p130 became the prominent RB protein. This suggests that RB2/P130 could be a marker of senescence or that it even plays a role in triggering the process in MSCs.