PURPOSE: Targeted treatments are desperately needed for ependymomas. Chimeric antigen receptor (CAR) T cells have immense potential to transform patient outcomes. However, CAR T-cell therapy for ependymomas has been largely understudied. In this study, we explore the potential of targeting B7 homolog 3 (B7-H3/CD276) with CAR T cells to treat pediatric ependymomas. EXPERIMENTAL DESIGN: We profiled B7-H3 protein expression in 44 pediatric ependymoma samples by IHC. We generated second-generation human B7-H3.CAR T cells and examined their anti-ependymoma activity in six in vitro and two in vivo xenograft models. We validated findings using HER2-targeted CAR T cells. In addition, we used murine B7-H3.CAR T cells to evaluate in vivo antitumor activity in a fully syngeneic supratentorial ependymoma model. RESULTS: The majority of clinical ependymoma samples (29/44) stained positive for B7-H3, indicating high but heterogeneous expression across patients. In vitro, human B7-H3.CAR T cells had potent anti-ependymoma cytolytic activity, expansion, and persistence, which was inversely correlated with the upregulation of B7-H3 on CAR T cells. We found that CAR T cells favor type 2 cytokines phenotypes after repeated exposure to ependymoma cell lines, which may be driven by C-C motif chemokine ligand 2 secretion by ependymomas. In vivo, there was potent and significant antitumor activity in human xenograft ependymoma models. However, response durability was limited and significantly correlated with the degree of tumor burden. In the syngeneic setting, murine B7-H3.CAR T-cell efficacy against ependymomas was limited and did not extend survival. CONCLUSIONS: Our results support ongoing clinical evaluation of B7-H3.CAR T cells for ependymomas and provide model systems for further studying determinants of anti-ependymoma CAR T-cell treatment efficacy and resistance.