While adoptive cell therapies have revolutionized cancer immunotherapy, current autologous chimeric antigen receptor (CAR) T cell manufacturing faces challenges in scaling to meet patient demands. CAR T cell production still largely relies on fed-batch, manual, open processes that lack environmental monitoring and control, whereas most perfusion-based, automated, closed-system bioreactors currently suffer from large footprints and working volumes, thus hindering process development and scaling-out. Here, we present a means of conducting anti-CD19 CAR T cell culture-on-a-chip. We show that human primary T cells can be activated, transduced, and expanded to densities exceeding 150 million cells/mL in a 2 mL perfusion-capable microfluidic bioreactor, thus enabling the production of CAR T cells at clinical dose levels in a small footprint. Key functional attributes such as exhaustion phenotype and cytolytic function were comparable to T cells generated in a gas-permeable well. The process intensification and miniaturization, together with the online analytics offered by the microbioreactor, could facilitate high-throughput process optimization studies, as well as enable efficient scale-out of cell therapy manufacturing, while providing insights into the growth and metabolic state of the CAR T cells during ex vivo culture.