Transforming growth factor-beta 1 (TGF-β1) stimulates a broad range of effects which are cell type dependent, and it has been suggested to induce cellular senescence. On the other hand, long-term culture of multipotent mesenchymal stromal cells (MSCs) has a major impact on their cellular physiology and therefore it is well conceivable that the molecular events triggered by TGF-β1 differ considerably in cells of early and late passages. In this study, we analyzed the effect of TGF-β1 on and during replicative senescence of MSCs. Stimulation with TGF-β1 enhanced proliferation, induced a network like growth pattern and impaired adipogenic and osteogenic differentiation. TGF-β1 did not induce premature senescence. However, due to increased proliferation rates the cells reached replicative senescence earlier than untreated controls. This was also evident, when we analyzed senescence-associated DNA-methylation changes. Gene expression profiles of MSCs differed considerably at relatively early (P 3-5) and later passages (P 10). Nonetheless, relative gene expression differences provoked by TGF-β1 at individual time points or in a time course dependent manner (stimulation for 0, 1, 4 and 12 h) were very similar in MSCs of early and late passage. These results support the notion that TGF-β1 has major impact on MSC function, but it does not induce senescence and has similar molecular effects during culture expansion.