ABSTRACT: The prognosis of Ewing sarcoma caused by EWS/FLI1 fusion is poor, especially after metastasis. Although therapy with cytotoxic T lymphocytes (CTLs) targeted against altered EWS/FLI1 sequences at the gene break/fusion site may be effective, CTLs generated from peripheral blood are often exhausted because of continuous exposure to tumor antigens. We addressed this by generating induced pluripotent stem cell (iPSC)-derived functionally rejuvenated CTLs (rejTs) directed against the neoantigen encoded by the EWS/FLI1 fusion gene. In this study, we examined the antitumor effects of EWS/FLI1-rejTs against Ewing sarcoma. The altered amino acid sequence at the break/fusion point of EWS/FLI1, when presented as a neoantigen, evokes an immune response that targets EWS/FLI1+ sarcoma. Although the frequency of generated EWS/FLI1-specific CTLs was only 0.003%, we successfully established CTL clones from a healthy donor. We established iPSCs from an EWS/FLI1-specific CTL clone and redifferentiated them into EWS/FLI1-specific rejTs. To evaluate cytotoxicity, we cocultured EWS/FLI1-rejTs with Ewing sarcoma cell lines. EWS/FLI1-rejTs rapidly and continuously suppressed the proliferation of Ewing sarcoma for >40 hours. Using an Ewing sarcoma xenograft mouse model, we verified the antitumor effect of EWS/FLI1-rejTs via imaging, and EWS/FLI1-rejTs conferred a statistically significant survival advantage. "Off-the-shelf" therapy is less destructive and disruptive than chemotherapy, and radiation is always desirable, particularly in adolescents, whom Ewing sarcoma most often affects. Thus, EWS/FLI1-rejTs targeting an Ewing sarcoma neoantigen could be a promising new therapeutic tool.