CD8+ T cells contribute to immune responses by producing cytokines when their T cell receptors (TCRs) recognise peptide antigens on major-histocompability-complex (pMHC) class I. However, excessive cytokine production can be harmful. For example, cytokine release syndrome (CRS) is a common toxicity observed in treatments that activate T cells, including chimeric antigen receptor (CAR)-T cell therapy. Whilst engagement of costimulatory receptors is well known to enhance cytokine production, we have limited knowledge of their ability to regulate the kinetics of cytokine production by CAR-T cells. Here we compare early (0-12 hours) and late (12-20 hours) production of IFN-gg, IL-2, and TNF-a production by T cells stimulated via TCR or CARs in the presence or absence ligands for CD2, LFA-1, CD28, CD27, and 4-1BB. For T cells expressing TCRs and 1st-generation CARs, activation by antigen alone was sufficient to stimulate early cytokine production, whilst co-stimulation by CD2 and 4-1BB was required to maintain late cytokine production. In contrast, T cells expressing 2nd-generation CARs, which have intrinsic costimulatory signalling motifs, produce high levels of cytokines in both early and late periods in the absence of costimulatory receptor ligands. Losing the requirement for costimulation for sustained cytokine production may contribute to the effectiveness and/or toxicity of 2nd-generation CAR-T cell therapy.