The development of inhibitory antibodies against factor VIII (FVIII) is the major complication in patients with haemophilia A who are treated with FVIII products. Memory B cells play an essential role in maintaining established antibody responses. Upon re-exposure to the same antigen, they are rapidly re-stimulated to proliferate and differentiate into antibody-secreting plasma cells (ASC) that secrete high-affinity antibodies. It is, therefore, reasonable to believe that memory B cells have to be eradicated or inactivated for immune tolerance induction therapy to be successful in patients with haemophilia A and FVIII inhibitors. The aim of our studies was the development of strategies to prevent FVIII-specific memory B cells from becoming re-stimulated. We established a 6-day in vitro culture system that enabled us to study the regulation of FVIII-specific murine memory-B-cell re-stimulation. We tested the impact of the blockade of co-stimulatory interactions, of different concentrations of FVIII and of ligands for toll-like receptors (TLR). The blockade of B7-CD28 and CD40-CD40 ligand interactions prevented FVIII-specific murine memory B cells from becoming re-stimulated by FVIII in vitro and in vivo. Furthermore, high concentrations of FVIII blocked re-stimulation of FVIII-specific murine memory B cells. Triggering of TLR7 amplified re-stimulation by low concentrations of FVIII and prevented blockade by high concentrations of FVIII. We conclude that we defined modulators that either amplify or inhibit the re-stimulation of FVIII-specific murine memory B cells. Currently, we are investigating whether the same modulators operate in patients with haemophilia A and FVIII inhibitors.