Abstract Engineered T-cell therapies have demonstrated impressive clinical responses in patients with hematologic malignancies. Despite this efficacy, many patients have a transient persistence of T cells, which can be correlated with transient clinical response. Translational data on T cells from pediatric cancer patients shows a progressive decline in chimeric antigen receptor (CAR) suitability with cumulative chemotherapy regardless of regimen. We investigated the effects of chemotherapy on surviving T cells in vitro, describing residual deficits unique to each agent including mitochondrial damage and metabolic alterations. In the case of cyclophosphamide but not doxorubicin or cytarabine, these effects could be reversed with N-acetylcysteine. Specifically, we observed that surviving T cells could be stimulated, expanded, and transduced with CARs with preserved short-term cytolytic function but at far lower numbers and with residual metabolic deficits. These data have implications for understanding the effects of chemotherapy on mature T cells later collected for adoptive cell therapy, as chemotherapy-exposed T cells may have lingering dysfunction that affects ex vivo adoptive cell therapy manufacturing techniques and, ultimately, clinical efficacy.