Circulating platelets are targeted by autoantibodies in various pathologic conditions, such as immune thrombocytopenic purpura (ITP). Anti-platelet antibodies cause thrombocytopenia through enhanced platelet clearance via Fcγ receptor-mediated platelet destruction by the reticuloendothelial system and impaired platelet production. Moreover, functional blockade of platelet surface receptors by autoantibodies may further promote bleeding tendency. The major targets of these autoantibodies are platelet membrane glycoproteins, including GPIIb/IIIa and GPIb/IX, receptors for fibrinogen and other platelet-activating ligands, but some patients with ITP have antibodies to a receptor for thrombopoietin, which is a growth factor required for megakaryocytogenesis and platelet production. Several antigen-specific assays have been developed to measure anti-glycoprotein antibodies, whereas we have recently established an enzyme-linked immunospot assay for the detection of circulating B cells secreting IgG anti-GPIIb/IIIa antibodies, which is a sensitive, specific, and convenient method for evaluating the presence or absence of ITP. Production of pathogenic anti-platelet antibodies is maintained by a continuous loop, in which B cells produce anti-platelet antibodies, antibody-coated platelets are phagocytosed and GPIIb/IIIa-derived cryptic peptides presented by splenic macrophages, and GPIIb/IIIa-reactive CD4⁺ T cells exert their helper activity. Important discoveries on cellular and molecular mechanisms for anti-platelet autoantibody production contribute to development of diagnostic assays and therapeutic strategies for ITP.