B-1 B cells derive from a developmental program distinct from that of conventional B cells, through B cell receptor (BCR)-dependent positive selection of fetally derived precursors. Here, we used direct labeling of B cells reactive with the N-acetyl-D-glucosamine (GlcNAc)-containing Lancefield group A carbohydrate of Streptococcus pyogenes to study the effects of bacterial antigens on the emergent B-1 B cell clonal repertoire. The number, phenotype, and BCR clonotypes of GlcNAc-reactive B-1 B cells were modulated by neonatal exposure to heat-killed S. pyogenes bacteria. GlcNAc-reactive B-1 clonotypes and serum antibodies were reduced in germ-free mice compared with conventionally raised mice. Colonization of germ-free mice with a conventional microbiota promoted GlcNAc-reactive B-1 B cell development and concomitantly elicited clonally related IgA+ plasma cells in the small intestine. Thus, exposure to microbial antigens in early life determines the clonality of the mature B-1 B cell repertoire and ensuing antibody responses, with implications for vaccination approaches and schedules. • GAC-reactive B cells exhibit the phenotype and localization patterns of B-1 B cells • Neonatal S. pyogenes immunization expands minor IGHV7-3 GAC-reactive BCR clonotypes • Establishment of IGHV6-3 GAC-reactive B cell clonal dominance is microbiota dependent • Microbiota-dependent expansion of GAC-reactive B cells seeds the SI LP with ASCs Innate-like B-1 B cells express canonical B cell receptors and originate through specialized developmental programs. Through immunoglobulin gene sequencing of single antigen-reactive B cells, New et al. demonstrate a significant role for environmental antigen-dependent selection events in shaping the GAC-reactive B-1 B cell clonal repertoire. [ABSTRACT FROM AUTHOR]