FVIII immunization increases the frequency of activated TFH cells in mice that produce inhibitors. FVIII^null mice were given 5 rounds of weekly IV FVIII immunizations. Five days after the last immunization, inhibitor titers were measured by Bethesda assay. Representative animals with inhibitors and no inhibitors were euthanized for TFH analysis. Age-matched naive FVIII^null mice were used as controls. Splenocytes were stained for CD3, CD4, CD19, CD44, CD62L, Foxp3, CXCR5, PD-1, and Ki-67, and analyzed by flow cytometry. (A) Percentage of effector helper cells (CD44⁺CD62L⁻) among Foxp3⁻CD4 T cells in saline-injected control mice, non-inhibitor–producing mice, and inhibitor-producing mice. (B) Percentage of CXCR5⁺PD-1⁺ TFH cells among effector CD4 helper cells. (C) Percentage of activated TFH cells among total CD4 T cells. (D) Total number of activated TFH cells per spleen. (E) Percentage of Ki-67⁺ in activated TFH cells. (F) Representative samples and summary data (n = 4 mice per group) of apoptotic cells in naive, non-TFH effector CD4 T cells and TFH cells from FVIII-immunized mice. *P < .05; **P < .01; ***P < .001.