Cells undergo replicative senescence during in vitro expansion, which is induced by the accumulation of cellular damage caused by excessive reactive oxygen species. In this study, we investigated whether long-term-cultured human bone marrow mesenchymal stromal cells (MSCs) are insensitive to apoptotic stimulation. To examine this, we established replicative senescent cells from long-term cultures of human bone marrow MSCs. Senescent cells were identified based on declining population doublings, increased expression of senescence markers p16 and p53 and increased senescence-associated β-gal activity. In cell viability assays, replicative senescent MSCs in late passages (i.e. 15–19 passages) resisted damage induced by oxidative stress more than those in early passages did (i.e. 7–10 passages). This resistance occurred via caspase-9 and caspase-3 rather than via caspase-8. The senescent cells are gradually accumulated during long-term expansion. The oxidative stress-sensitive proteins ataxia-telangiectasia mutated and p53 were phosphorylated, and the expression of apoptosis molecules Bax increased, and Bcl-2 decreased in early passage MSCs; however, the expression of the apoptotic molecules did less change in response to apoptotic stimulation in late-passage MSCs, suggesting that the intrinsic apoptotic signalling pathway was not induced by oxidative stress in long-term-cultured MSCs. Based on these results, we propose that some replicative senescent cells may avoid apoptosis signalling via impairment of signalling molecules and accumulation during long-term expansion. Copyright © 2016 John Wiley & Sons, Ltd.