Periodic fever, aphthous stomatitis, pharyngitis, and adenitis (PFAPA) syndrome is a pediatric immune disorder with unknown pathogenesis that is characterized by recurrent fevers of fixed periodicity, without evidence of infection. Tonsillectomy has been shown to be curative. We hypothesize that PFAPA patients have a dysregulated innate immune system that results in the predictable flares. To characterize the innate immune mechanisms underlying PFAPA syndrome, we evaluated patient-derived tonsillar cells ex vivo and in vitro. Ex vivo analysis demonstrated increased IL-1 receptor antagonist (IL1RN) and tumor necrosis factor (TNF) gene expression in PFAPA tonsils, with persistent phosphorylation of IRF and NF-kappaB pathways. Febrile effects in vitro suggest an autoinflammatory feedback loop contributes to the pathogenesis behind PFAPA. Our results suggest that PFAPA patients experience ongoing subclinical inflammation in the tonsillar microenvironment, even during asymptomatic periods, and provide a mechanism for the success of tonsillectomy in this disease. The unique cytokine signature detected in the tonsil samples supports our recognition of PFAPA as an autoinflammatory disorder, but suggests the underlying pathology is of greater complexity than the previously described monogenic disorders. In addition, the distinct expression of cytokines in PFAPA tonsils compared to controls suggests that measurement of IL1RN and TNF expression, as performed in this study or in saliva may be a novel biomarker for the diagnosis of PFAPA.