Systemic lupus erythematosus (SLE) is an autoimmune disease with a high incidence in females and a complex phenotype. Using 564Igi mice, a model of SLE with knock-in genes encoding an autoreactive anti-RNA Ab, we investigated how expression of Toll-like receptors (TLRs) in B cells and neutrophils affects pathogenesis. We established that TLR signaling through MyD88 is necessary for disease. Autoantibody was produced in mice with single deletions of Tlr7, Tlr8, or Tlr9 or combined deletions of Tlr7 and Tlr9. Autoantibody was not produced in the combined absence of Tlr7 and Tlr8, indicating that TLR8 contributes to the break in tolerance. Furthermore, TLR8 was sufficient for the loss of B-cell tolerance, the production of class-switched autoantibody, heightened granulopoiesis, and increased production of type I IFN by neutrophils as well as glomerulonephritis and death. We show that dosage of X-linked Tlr8 plays a major role in the high incidence of disease in females. In addition, we show that the negative regulation of disease by TLR9 is exerted primarily on granulopoiesis and type I IFN production by neutrophils. Collectively, we suggest that individual TLRs play unique roles in the pathogenesis of systemic lupus erythematosus, suggesting new targets for treatment. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.