Chimeric antigen receptor (CAR) T cell therapies using the 28ζ CAR has shown promising therapeutic responses against hematological malignancies, but it has limitations in treatments against solid tumors. A major cause for poor outcomes in solid tumors is decreased CAR-T cell persistence, which may be linked to high tonic signaling. Tonic signaling is a process in which CAR-T cells activate spontaneously in the absence of tumor antigen stimulation. Previous studies have indicated that both the CD28 costimulatory domain and CD3ζ signaling domain contribute to tonic signaling in the 28ζ CAR, and that manipulating the type and number of the domains may optimize tonic signaling and improve CAR function. In this study, I assessed the tonic signaling phenotypes and cytotoxic potential of the anti-mesothelin 28ζ CAR-T cell and several single-ITAM CD3ζ-truncate mutants of the 28ζ CAR. I showed that the single-ITAM CARs showed more sustained tonic signaling contributing to enhanced growth without antigen but also earlier exhaustion. However, the 28ζ ITAM-mutants still showed comparable cytotoxicity to the wild-type 28ζ CAR in vitro.