Epstein-Barr virus (EBV) is an aetiologic risk factor for the development of multiple sclerosis (MS). However, the role of EBV-infected B cells in the immunopathology of MS is not well understood. Here we characterized spontaneous lymphoblastoid cell lines (SLCLs) isolated from MS patients and healthy controls (HC) ex vivo to study EBV and host gene expression in the context of an individual's endogenous EBV. SLCLs derived from MS patient B cells during active disease had higher EBV lytic gene expression than SLCLs from MS patients with stable disease or HCs. Host gene expression analysis revealed activation of pathways associated with hypercytokinemia and interferon signalling in MS SLCLs and upregulation of forkhead box protein 1 (FOXP1), which contributes to EBV lytic gene expression. We demonstrate that antiviral approaches targeting EBV replication decreased cytokine production and autologous CD4 T cell responses in this ex vivo model. These data suggest that dysregulation of intrinsic B cell control of EBV gene expression drives a pro-inflammatory, pathogenic B cell phenotype that can be attenuated by suppressing EBV lytic gene expression.