A pivotal role for tertiary lymphoid structures (TLSs) in promoting Ag-specific humoral responses during chronic inflammation is emerging in several autoimmune conditions, including rheumatoid arthritis, Sjogren's syndrome, and autoimmune thyroiditis. However, there is limited evidence on the cellular and molecular mechanisms underlying TLS formation and their contribution to autoimmunity in the pancreas during autoimmune insulitis. In this study, we performed a detailed and comprehensive assessment of the evolution of TLSs during autoimmune insulitis in 126 female NOD mice from 4 to 38 wk of age. We demonstrated that during progression from peri- to intrainsulitis in early diabetic mice, T and B cell infiltration follows a highly regulated process with the formation of lymphoid aggregates characterized by T/B cell segregation, follicular dendritic cell networks, and differentiation of germinal center B cells. This process is preceded by local upregulation of lymphotoxins alpha/beta and lymphoid chemokines CXCL13 and CCL19, and is associated with infiltration of B220(+)/IgD(+)/CD23(+)/CD21(-) follicular B cells expressing CXCR5. Despite a similar incidence of insulitis, late diabetic mice displayed a significantly reduced incidence of fully organized TLSs and reduced levels of lymphotoxins/lymphoid chemokines. Upon development, TLSs were fully functional in supporting in situ autoreactive B cell differentiation, as demonstrated by the expression of activation-induced cytidine deaminase, the enzyme required for Ig affinity maturation and class switching, and the presence of CD138(+) plasma cells displaying anti-insulin reactivity. Overall, our work provides direct evidence that TLSs are of critical relevance in promoting autoimmunity and chronic inflammation during autoimmune insulitis and diabetes in NOD mice.