We previously identified IgM autoantibodies in the sera of patients with Wiskott‐Aldrich syndrome (WAS) that react with a subset of normal human B lymphocytes and induce B cell differentiation in vitro. From splenocytes of a patient with WAS we generated heterohybridomas (HY18 and HY21) and a lymphoblastoid cell line (LWA10) that produce human IgM λ or IgM χ anti‐B lymphocyte autoantibodies, respectively. Immunohistochemical and multiparameter flow cytometric analyses demonstrate that these autoantibodies are specific for lymphocytes of the B lineage and preferentially stain B cells that reside in the mantle zone of secondary follicles and that constitutively co‐express the CD5 surface antigen and most major autoantibody‐associated cross‐reactive idiotypes; in addition, these antibodies stain most pre‐B cells in adult bone marrow. Molecular studies show that these anti‐B lymphocyte autoantibodies are encoded by a highly conserved V_H4 gene, designated V_H4.21. The gene encodes a number of autoantibodies, especially anti‐i and anti‐I IgM cold agglutinins. Hemagglutination and surface labeling studies reveal that HY18 and LWA10 recognize the “i” carbohydrate antigenic determinant(s) which is classically found on human cord red blood cells and, as shown now by this study, on a subpopulation of human B cells which expresses it early in B cell development. These studies raise the possibility that the gene product encoded by this highly conserved germ‐line V_H4 gene may play a physiological role in B cell development and/or differentiation.