Peripheral tolerance is partially controlled by ectopic peripheral tissue antigen (PTA) expression in lymph node stromal cells. We previously showed that the transcriptional regulator deformed epidermal autoregulatory factor 1 (Deaf1) can regulate the transcription, processing and/or presentation of PTA genes in lymph node stromal cells of the pancreatic lymph nodes (PLN). However, during the progression of diabetes, Deaf1 is spliced in the PLN to form a dominant negative isoform, Deaf1-Var. Expression of this isoform correlates with the severity of disease, suggesting that both inflammation and hyperglycemia may induce the splicing of Deaf1. We showed that Deaf1-Var expression is increased in the inflamed PLN of mice after i.p. injection of activated splenocytes that home to the PLN, and in the hyperglycemic PLN of mice after treatment with the insulin receptor antagonist S961. By microarray and QPCR analysis, we identified several genes (Ptbp2, Srsf10, and Ddx42) that may be involved in the splicing of Deaf1. Interestingly, PTBP2 was also found to be upregulated in the PLN of prediabetic autoantibody positive individuals compared to non-diabetic control patients. Together, these data suggest that inflammation and hyperglycemia that occur in the PLN during the progression of Type 1 diabetes lead to upregulation of various splicing factors that mediate Deaf1 splicing. This results in loss of Deaf1 function and reduced PTA expression in lymph node stromal cells.