ABSTRACT: Although clinical responses with CD19 targeting CAR T-cell treatment have been observed in patients with certain hematological malignancies, high rates of disease relapse highlight the necessity to understand and improve mechanisms of CAR T-cell failure. Since T-cell dysfunction is thought to contribute to CAR T-cell treatment failure, understanding what mechanisms drive T cells into this dysfunctional state may aid optimal design of efficacious CAR T cells. Dysfunctional CAR T cells have been characterized as having upregulated inhibitory receptors and decreased cytolytic capabilities. Previous studies have identified a role for sustained CAR CD3zeta signaling in CAR T-cell dysfunction. Here, we demonstrate a mechanism that drives dysfunction in CAR T cells through excessive costimulation. Fully activated CD19 targeted CAR T cells were rendered dysfunctional upon stimulation with both endogenous CD28 stimulation and CAR mediated CD28 costimulation. Costimulation driven dysfunction of CAR T cells was demonstrated in a syngeneic immunocompetent mouse model, in which CAR T cells were activated with signals 1 (CD3zeta), 2 (CD28), and 3 (IL12). Thus, we show that CAR T-cell dysfunction can be driven through excessive CD28 and 4-1BB costimulation.