Background: Treatment of B cell malignancies with CAR-modified T cells has shown remarkable efficacy, but clinical responses in solid tumors have been limited due to poor T cell persistence and expansion. We examined whether incorporation of unconventional signaling elements, derived from the “universal” toll-like receptor adaptor molecule, MyD88, and the TNF family member, CD40, into CARs could improve T cell survival, proliferation and antitumor efficacy. Methods: Bicistronic vectors encoding inducible Caspase-9 (iCasp9) and CD19- or Her2-targeted CARs incorporating various costimulatory domains (e.g., CD28, 4-1BB, MyD88/CD40) were generated and used to transduce T cells. CAR-T cells were assessed for antitumor function in tumor coculture assays using CD19⁺ (Raji and Daudi) cells or Her2⁺ (SK-BR-3) tumor cells. Efficacy was monitored in immunodeficient (NSG) mice engrafted with tumor cell lines followed by i.v. CAR-T cell injection. In vivo T cell proliferation, as well as elimination via iCasp9 after i.p. injection of rimiducid, was measured using bioluminescent imaging (BLI). Tumor burden was also assessed by BLI or caliper measurements. Results: All CAR constructs could be stably expressed in T cells (30-90% CAR expression). MC costimulation resulted in greatly increased IL-2 production compared to CARs with alternative costimulation (~2500 pg/mL and ~200 pg/mL for MC and CD28.41BB, respectively). This correlated with enhanced T cell proliferation and corresponded to better tumor elimination in coculture assays (99% elimination at a 1:1 E:T ratio within 14 days). MC costimulation also enhanced the efficacy of CAR-T therapy in CD19⁺ and Her2⁺ tumor models, respectively, compared to control CARs. Furthermore, T cells transduced with MC-encoding CARs could be rapidly eliminated in vivo (within 1 day) via iCasp9 following rimiducid administration. Conclusions: MyD88/CD40 represents a potent, new T cell costimulatory molecule for CAR-T cells, resulting in robust IL-2 production, improved survival and proliferation, increased antitumor activity against CD19⁺ and Her2⁺ tumors in vitro and in vivo, while retaining a high safety profile through the iCasp9 suicide gene.