Abstract Therapeutic B cell depletion in mice using CD20 mAb significantly reduces CD4+ T cell activation in response to model and self-antigens, but not CD8+ T cell activation. However, the effects of B cell depletion on acute viral infections and the consequences of long-term B cell depletion on immune system function are unknown. To assess this, B cells were depleted in wild type mice using a potent CD20 mAb that depletes >98% of tissue and circulating mature B cells. Short-term (2 week) B cell depletion did not affect tissue T cell numbers. However, when B cells were depleted 1 week before LCMV-Armstrong infection, splenic CD44hiCD62Llo CD4+ and CD8+ effector-memory T cell numbers were reduced by 51-57%. The numbers of LCMV-specific CD4+ T cells producing IFNγ and TNFα were reduced by 64% and 80%, respectively. Virus-specific CD8+ T cells producing IFNγ and TNFα were also reduced by >56%. Even without pathogen challenge, similar changes were observed in mice treated chronically with CD20 mAb for 6 months, where CD4+ and CD8+ effector-memory T cell numbers were reduced by 76% and 39%, respectively. Chronic B cell depletion also reduced IFNγ- and TNFα-producing CD4+ T cell numbers by 76% and 35%. Nonetheless, naïve T cell numbers were not affected by either short-term or chronic B cell depletion. Thus, B cells are required for optimal CD4+ and CD8+ T cell activation, expansion, and function in vivo, particularly during cellular responses to pathogens.