Autoimmunity and inflammation are controlled in part by regulatory B cells, including a recently identified IL-10-competent CD1d(high)CD5(+) B cell subset termed B10 cells that represents 1-3% of adult mouse spleen B cells. In this study, pathways that influence B10 cell generation and IL-10 production were identified and compared with previously described regulatory B cells. IL-10-competent B cells were predominantly CD1d(high)CD5(+) in adult spleen and were the prevalent source of IL-10, but not other cytokines. B10 cell development and/or maturation in vivo required Ag receptor diversity and intact signaling pathways, but not T cells, gut-associated flora, or environmental pathogens. Spleen B10 cell frequencies were significantly expanded in aged mice and mice predisposed to autoimmunity, but were significantly decreased in mouse strains that are susceptible to exogenous autoantigen-induced autoimmunity. LPS, PMA, plus ionomycin stimulation in vitro for 5 h induced B10 cells to express cytoplasmic IL-10. However, prolonged LPS or CD40 stimulation (48 h) induced additional adult spleen CD1d(high)CD5(+) B cells to express IL-10 following PMA plus ionomycin stimulation. Prolonged LPS or CD40 stimulation of newborn spleen and adult blood or lymph node CD1d(low) and/or CD5(-) B cells also induced cytoplasmic IL-10 competence in rare B cells, with CD40 ligation uniformly inducing CD5 expression. IL-10 secretion was induced by LPS signaling through MyD88-dependent pathways, but not following CD40 ligation. LPS stimulation also induced rapid B10 cell clonal expansion when compared with other spleen B cells. Thereby, both adaptive and innate signals regulate B10 cell development, maturation, CD5 expression, and competence for IL-10 production.