The development of neutralizing antibodies (inhibitors) after factor VIII (FVIII) infusions is a serious complication that affects approximately one-quarter of hemophilia A patients who have access to replacement therapy. To investigate the differentiation of naive T cells into FVIII-specific helper T cells that promote B-cell activation and antibody secretion, HLA-DRA-DRB1*0101-restricted T-cell clones that respond to a specific epitope in FVIII were isolated from a mild hemophilia A subject (the proband) 19 weeks and 21 months after his development of a high-titer inhibitor. Clones responding to the same epitope were also isolated from his multiply infused brother, who has not developed a clinically significant inhibitor. The 19-week proband clones were T helper (T(H))17/T(H)1- or T(H)1/T(H)2-polarized, whereas all 8 clones isolated 21 months postinhibitor development were T(H)2-polarized cells. In contrast, all 6 clones from the brother who did not develop an inhibitor were T(H)1-polarized, indicating that tolerance to FVIII can be maintained even with circulating T(H)1-polarized cells that respond vigorously to in vitro FVIII stimulation. This is the first evidence that T(H)17/T(H)1-polarized cells play a role in hemophilic immune responses to FVIII. Furthermore, this is the first report of successful isolation and expansion of antigen-specific human T(H)17/T(H)1 clones using standard culture conditions.