Using the murine model of hemophilia A, we have examined the role of CD154 in the secondary immune response to factor VIII (FVIII). We previously reported that repeated i.v. injection of FVIII in hemophilia A mice induces a T cell‐dependent anti‐FVIII antibody formation. Herein, blocking of CD154 by a monoclonal antibody in FVIII‐primed hemophilia A mice resulted in the disappearance of pre‐existing spleen germinal centers (GC) in the white pulp within 24 h of treatment. Moreover, further expansion of GC in response to FVIII challenge was completely inhibited. In parallel, anti‐FVIII antibody titers were markedly reduced and T cell responses to FVIII were abolished. The rapid disappearance of the GC after anti‐CD154 treatment was not accompanied by increased B cell apoptosis; instead B cells accumulated in the peripheral zone of the splenic white pulp. Interestingly, repeated exposure to FVIII with anti‐CD154 antibody administration blocked anti‐FVIII antibody formation but failed to induce long‐lasting unresponsiveness. Our data demonstrate that the CD40‐CD154 interaction is critical for B cell homeostasis and the secondary immune response to FVIII. For potential clinical application, the data also suggest that therapies targeting the CD154 molecule may be useful for the treatment of high titer FVIII inhibitors in hemophilia A.