Abstract Checkpoint blockade treatment has shown great potential in both chronic viral infection and cancer patients, however the efficacy relies on the number of responsive CD8 T cells. Recently a population of CXCR5+ follicular CD8 T cells (TFC) has been identified in chronic LCMV infection and cancer models as the cells provide the proliferation burst and effector function following anti-PD-1/PD-L1 therapy. Understanding the cellular and molecular mechanisms regulate TFC differentiation and expansion will be crucial for improving the efficacy of immunotherapy. Here, we demonstrate that blockade of IFN-I signaling promotes a cell-intrinsic and sustained generation of TFC following persistent virus infection. Paradoxically, we observed a subset of ISG in TFC were induced by the cytokine IL-27 via STAT1 transcriptional program in the absence of IFN-I signaling. Moreover, blockade of IFN-I signaling either with antibody or pharmacologically through JAK inhibition, promoted the expansion of TFC during viral infection through an IL-27 and STAT1-dependent mechanism. Our results uncover a contrasting role of IL-27 and IFN type I in regulating the expansion of TFC despite their highly overlapping transcriptional signatures. Moreover, we demonstrate that pharmacological inhibition of JAK1/2 signaling induces optimal TFC generation without compromise effector CD8 T cell proliferation and function.