Chimeric antigen receptor T cell (CAR-T) therapies show high remission rates for relapsed and refractory leukemia and lymphoma. However, manufacturing challenges hinder their commercial viability and patient accessibility. This study applied quality-by-design principles to identify perfusion critical process parameters for CAR-T expansion in stirred tank bioreactors to maximize yields. A design of experiments in the Ambr® 250 High Throughput Perfusion small-scale bioreactor revealed that earlier perfusion starts (48 h vs. 96 h post-inoculation) and higher perfusion rates (1.0 VVD vs. 0.25 VVD) significantly increased cytotoxic CAR-T cell yields without compromising critical quality attributes. Optimizing perfusion improved growth kinetics and yields across donor samples, achieving densities >21 × 10 cells/mL in 7 days, outperforming traditional fed-batch and static flask cultures. This study underscores the importance of optimizing perfusion parameters to maximize CAR-T yields and quality and highlights the utility of scale-down models in reducing time, costs and risks associated with process development.