Immune complexes consisting of heparin, platelet factor 4 (PF4), and PF4/heparin-reactive antibodies are central to the pathogenesis of heparin-induced thrombocytopenia (HIT). It is as yet unclear what triggers the initial induction of pathogenic antibodies. We identified B cells in peripheral blood of healthy adults that produce PF4/heparin-specific antibodies following in vitro stimulation with proinflammatory molecules containing deoxycytosine-deoxyguanosine (CpG). Similarly, B cells from unmanipulated wild-type mice produced PF4/heparin-specific antibodies following in vitro or in vivo CpG stimulation. Thus, both healthy humans and mice possess preexisting inactive/tolerant PF4/heparin-specific B cells. The findings suggest that breakdown of tolerance leads to PF4/heparin-specific B-cell activation and antibody production in patients developing HIT. Consistent with this concept, mice lacking protein kinase Cδ (PKCδ) that are prone to breakdown of B-cell tolerance produced anti-PF4/heparin antibodies spontaneously. Therefore, breakdown of tolerance can lead to PF4/heparin-specific antibody production, and B-cell tolerance may play an important role in HIT pathogenesis.