Restimulation and differentiation of FVIII-specific memory B cells requires FVIII and interaction with activated T cells. Memory cell pool cells (CD138^–T⁺) and memory cell pool cells depleted of T cells (CD138^–T^–) were obtained from spleen cells of hemophilic mice treated with 4 intravenous doses of 200 ng (80 U/kg) FVIII. (A) CD138^–T⁺ cells, CD138^–T^– cells, and CD138^–T^– cells supplemented with T cells from either naive mice (naive T cells), mice treated with 4 intravenous doses of FVIII (FVIII T cells), or mice treated with 3 intraperitoneal doses of ovalbumin (Ova T cells) were cultured in the presence of either FVIII alone, FVIII plus ovalbumin (FVIII + OVA), or ovalbumin alone (OVA). CD138^–T^– and T cells were always mixed at a ratio of 1:2. Newly formed ASCs were analyzed by using ELISPOT assays after 6 days of culture. (B) Ovalbumin-sensitized mice received memory cell pool cells depleted of T cells and were challenged 1 day after transfer with either ovalbumin together with FVIII (▵) or ovalbumin alone (○). For comparison, naive mice received memory cell pool cells (▪) or memory cell pool cells depleted of T cells (⋄) and were challenged with FVIII 1 day after cell transfer. Blood samples were obtained 7, 14 and 21 days after treatment and analyzed for anti-FVIII antibodies by ELISA. Each point represents a single mouse.