Abstract Hemophilia A patients lack functional factor VIII (FVIII), which is essential for normal blood clotting. Approximately 1 in 4 patients receiving replacement therapy by infusions of FVIII develop neutralizing antibodies that can cause serious bleeds. Tetramer-guided epitope mapping (TGEM) experiments identified an HLA-DRB1*01:01-restricted T-cell epitope in 2 subjects with missense substitution A2201P. The same epitope was identified using samples from a subject with severe hemophilia. T-cell clones were isolated by single-cell sorting and expansion of tetramer-positive CD4 T cells. Peptide-MHC binding assays using recombinant DR0101 and FVIII peptides with systematic arginine substitutions identified anchor residues F2196, M2199, A2201 and S2204. Recombinant FVIII-C2 domain with substitution F2196A abrogated proliferation of 4 clones. Six FVIII proteins with rational substitutions designed to interfere with epitope presentation (F2196A, F2196K, F2196L, M2199A, M2199W and M2199R) were produced in a BHK expression system. All had pro-coagulant activities, measured by both clotting and chromogenic assays, similar to that of wild-type FVIII. Their immunogenicity is now being evaluated. These experiments provide proof of principle for the design of less immunogenic, functional FVIII proteins targeted to specific subsets of patients.