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Chimeric antigen receptor (CAR) T cells are highly effective in haematological malignancies. However, progressive loss of CAR T cells contributes to relapse in many patients. Here we performed in vivo loss-of-function CRISPR screens in CAR T cells targeting B cell maturation antigen to investigate genes that influence CAR T cell persistence and function in a human multiple myeloma model. We tracked the expansion and persistence of CRISPR library-edited T cells in vitro and at early and late time points in vivo to track the performance of gene-modified CAR T cells from manufacturing to survival in tumours. The screens revealed context-specific regulators of CAR T cell expansion and persistence. Ablation of RASA2 and SOCS1 enhanced T cell expansion in vitro, whereas loss of PTPN2, ZC3H12A and RC3H1 conferred early growth advantages to CAR T cells in vivo. Notably, we identified cyclin-dependent kinase inhibitor 1B (encoded by CDKN1B), a cell cycle regulator, as the most important factor limiting CAR T cell fitness at late time points in vivo. CDKN1B ablation increased CAR T cell proliferation and effector function, significantly enhancing tumour clearance and overall survival. Our findings reveal differing effects of gene perturbation on CAR T cells over time and in different environments, highlight CDKN1B as a promising target to generate highly effective CAR T cells for multiple myeloma and underscore the potential of in vivo screening for identifying genes to enhance CAR T cell efficacy.