Mutations of the Wiskott-Aldrich syndrome gene (WAS) are responsible for the Wiskott-Aldrich syndrome, a disease characterized by thrombocytopenia, eczema, immunodeficiency and autoimmunity. Mice with conditional deficiency of Was in B lymphocytes (B/WcKO) have revealed a critical role for WAS protein (WASP) expression in B lymphocytes in the maintenance of immune homeostasis. Neural WASP (N-WASP) is a broadly expressed homologue of WASP, and regulates signaling in B cells by modulating B cell receptor (BCR) clustering and internalization. To investigate whether N-WASP expression in B cells plays a role in the development of autoimmunity in WAS, we have generated a double conditional mouse lacking both WASP and N-WASP selectively in B lymphocytes (B/DcKO mouse). As compared to B/WcKO mice, B/DcKO mice showed defective B lymphocyte proliferation in response to simultaneous stimulation via BCR and TLR9, and impaired antibody responses to T cell-dependent antigens. Defective B cell activation in B/DcKO mice was associated with decreased autoantibody production and lack of autoimmune kidney disease, as compared to B/WcKO mice. These results demonstrate that N-WASP expression in B lymphocytes is required for the development of autoimmunity of WAS and may represent a novel therapeutic target in this disease.