After long-term culture, mesenchymal stem cells alter their biological properties and enter into a state of replicative senescence. Although several classical biomarkers have been used for quantitative assessment of cellular senescence, no hallmark has been proven completely unique to the senescent state in cells. We used bone marrow-derived MSCs (BM-MSCs) from different healthy young donors and an in vitro model with well-defined senescence end points to identify a set of robust markers that could potentially predict the expansion capacity of MSCs preparations before reaching senescence. For each early passage BM-MSC sample (5th or 6th passages), the normalized protein expression levels of senescence-associated markers p16(INK4A), p21(WAF1), SOD2, and rpS6(S240/244); the concentration of IL6 and IL8 in cell culture supernatants; and the normalized gene expression levels of pluripotency markers OCT4, NANOG, and SOX2 were correlated with final population doubling (PD) number. We revealed that the low expression of p16(INK4A) protein and a high OCT4 gene expression, rather than other evaluated markers, might be potential hallmarks and predictors of greater in vitro lifespan and growth potential, factors that can impact the successful therapeutic use of MSCs preparations.