Hemophilia A (coagulation factor FVIII deficiency), inherited as an X-linked recessive trait, is the most prevalent genetic bleeding disorder worldwide (1 in 5000 male births). Conventional treatment involves intravenous replacement therapy with recombinant or plasma derived clotting factor, which poses a risk for developing anti-drug antibodies (inhibitors) in up to 30% of severe patients. Inhibitors largely neutralize the infused replacement clotting factor, thus limiting availability, increase the risk for morbidity and mortality and may also cause immunotoxicities. Immune tolerance induction to eradicate inhibitors has a significant failure rate. Inhibitor development is CD4+T helper cell dependent and tolerance via recruitment of regulatory T cells (Tregs) represents a potential approach to control inhibitor development. Here we evaluated cellular therapy with antigen-specific Tregs engineered to express a murine 2ndgeneration chimeric antigen receptor (CAR) specific to human FVIII. FVIII CAR-Tregs were activated and proliferated in response to soluble recombinant hFVIII, secreting key cytokines in vitro. Activation induced cell death (AICD) was observed in a significant percent of stimulated Teff cells, while Tregs were more resistant to AICD. Introducing specific mutations in immunoreceptor tyrosine-based activation motifs (ITAMs) in CD3z was able to improve AICD. Ex vivo expanded FVIII CAR-Tregs, when adoptively transferred into mice with a targeted deletion in exon 16 that rendered them deficient in endogenous FVIII (BALB/ce16-/-hemophilia A mice), prevented the formation of inhibitors. Studies are ongoing to test whether cellular therapy with FVIII CAR-Tregs can tolerize mice that have already established inhibitors, which would more closely model clinical disease in patients.