The development of neutralizing anti-FVIII antibodies (inhibitors) is a major complication of FVIII protein replacement therapy in patients with hemophilia A (HA). Although multiple lines of evidence indicate that the immune response against FVIII is CD4 T-cell–dependent and many FVIII-derived CD4 epitopes have already been discovered, the role of T follicular helper (TFH) cells in FVIII inhibitor development is unknown. TFH cells, a newly identified subset of CD4 T cells, are characterized by expression of the B-cell follicle-homing receptor CXCR5 and PD-1. In this study, we show for the first time that IV FVIII immunization induces activation and accumulation and/or expansion of PD-1⁺CXCR5⁺ TFH cells in the spleen of FVIII-deficient (FVIII^null) mice. FVIII inhibitor-producing mice showed increased germinal center (GC) formation and increased GC TFH cells in response to FVIII immunization. Emergence of TFH cells correlated with titers of anti-FVIII inhibitors. Rechallenge with FVIII antigen elicited recall responses of TFH cells. In vitro FVIII restimulation resulted in antigen-specific proliferation of splenic CD4⁺ T cells from FVIII-primed FVIII^null mice, and the proliferating cells expressed the TFH hallmark transcription factor BCL6. CXCR5^+/+ TFH-cell–specific deletion impaired anti-FVIII inhibitor production, confirming the essential role of CXCR5^+/+ TFH cells for the generation of FVIII-neutralizing antibodies. Together, our results demonstrate that the induction of activated TFH cells in FVIII^null mice is critical for FVIII inhibitor development, suggesting that inhibition of FVIII-specific TFH-cell activation may be a promising strategy for preventing anti-FVIII inhibitor formation in patients with HA.