Adipose-derived stem cells (ADSCs) have led to growing interest in cell-based therapy because they can be easily harvested from an abundant tissue. ADSCs must be expanded in vitro before transplantation. This essential step causes concerns about the safety of adult stem cells in terms of potential transformation. Tumorigenesis is driven in its earliest step by DNA replication stress, which is characterized by the accumulation of stalled DNA replication forks and activation of the DNA damage response. Thus, to evaluate the safety of ADSCs during ex vivo expansion, we monitored DNA replication under atmospheric (21%) or physiologic (1%) oxygen concentration. Here, by combining immunofluorescence and DNA combing, we show that ADSCs cultured under 21% oxygen accumulate endogenous oxidative DNA lesions, which interfere with DNA replication by increasing fork stalling events, thereby leading to incomplete DNA replication and fork collapse. Moreover, we found by RNA sequencing (RNA-seq) that culture of ADSCs under atmospheric oxygen concentration leads to misexpression of cell cycle and DNA replication genes, which could contribute to DNA replication stress. Finally, analysis of acquired small nucleotide polymorphism shows that expansion of ADSCs under 21% oxygen induces a mutational bias toward deleterious transversions. Overall, our results suggest that expanding ADSCs at a low oxygen concentration could reduce the risk for DNA replication stress-associated transformation, as occurs in neoplastic tissues. SIGNIFICANCE:The present work explored the influence of oxygen concentration on the induction of DNA replication stress, a major driver of tumorigenesis, during ex vivo amplification of adipose-derived stem cells (ADSCs) used for cellular therapies. The currently used good manufacturing practices atmospheric oxygen condition (21%) compared with the physiologic one (1%) increased oxidative DNA damage and perturbed DNA replication by blocking DNA replication forks and, as a result, enhanced transversion mutations and activated the DNA damage response pathways. Collectively, these results strongly suggest that decreasing oxygen concentration could improve the safety of ADSCs during ex vivo expansion.