To interrogate the role of specific immune cells in infection, cancer, and autoimmunity, immunologists commonly use monoclonal depletion antibodies (depletion-mAbs) or genetically engineered mouse models (GEMMs). To generate a tool that combines specific advantages and avoids select drawbacks of the two methods, we engineered adeno-associated viral vectors expressing depletion mAbs (depletion-AAVs). Single-dose depletion-AAV administration durably eliminated lymphocyte subsets in mice and avoided accessory deficiencies of GEMMs, such as marginal zone defects in B cell-deficient animals. Depletion-AAVs can be used in animals of different genetic backgrounds, and multiple depletion-AAVs can readily be combined. Exploiting depletion-AAV technology, we showed that B cells were required for unimpaired CD4⁺ and CD8⁺ T cell responses to chronic lymphocytic choriomeningitis virus (LCMV) infection. Upon B cell depletion, CD8⁺ T cells failed to suppress viremia, and they only helped resolve chronic infection when antibodies dampened viral loads. Our study positions depletion-AAVs as a versatile tool for immunological research.