A large body of evidence supports the role of activated stromal cells in the persistence of inflammation. The switch from resting to pathogenic stroma appears to be associated with the development of tertiary lymphoid organs (TLOs) within sites of chronic inflammation. However little is known about the immunological function of the stromal component. We utilised a murine model of inducible TLO formation in inflamed salivary glands to investigate the role of activated stromal cells characterised by the expression of gp38 and FAP during TLO development. We demonstrated that during inflammation, stroma-derived ICOSL engages ICOS on T cells, necessary for the release of lymphotoxin α and consequent TLO formation. Whilst dissecting the role of stromal cells in this context, we demonstrate that gp38 expression is required for the upregulation of adhesion molecules involved in cell clustering. Depletion of gp38+FAP+ stromal cells led to a significant reduction in lymphoid chemokine production, a decreased number of infiltrating lymphocytes and severely compromised TLO formation. Collectively, we provide evidence that activated stromal cells express FAP, provide co-stimulatory signals, and are necessary for the establishment of viral-induced TLOs, highlighting a potential novel therapeutic target in TLO-associated autoimmune diseases.