Chimeric antigen receptors (CARs) are engineered proteins designed to target T cells to cancer cells. To effectively activate the T cells in which they are expressed, CARs must contain a costimulatory domain. The CAR T cell products approved for the treatment of B cell lymphomas and/or acute lymphoblastic leukaemia or multiple myeloma incorporate either a CD28-derived or a 4-1BB-derived costimulatory domain. Almost all other clinically tested CARs also use costimulatory domains from CD28 or 4-1BB. In preclinical experiments, cytokine release is usually greater with CARs containing CD28 versus 4-1BB costimulatory domains; however, constructs with either domain confer similar anticancer activity in mouse models. T cell products expressing CARs with either CD28 or 4-1BB costimulatory domains have been highly efficacious in patients with relapsed haematological malignancies, with anti-CD19 products having similar activity regardless of the source of the costimulatory domain. In large-cohort clinical trials, the rates of neurological toxicities have been higher with CD28-costimulated CARs, although this finding is probably the result of a combination of factors rather than due to CD28 signalling alone. Future preclinical and clinical research should aim to compare different costimulatory domains while controlling for confounding variables. Herein, we provide an overview of T cell costimulation by CD28 and 4-1BB and, using the available preclinical and clinical data, compare the efficacy and toxicity profiles associated with CARs containing either costimulatory domain.