Early viral infection is often associated with lymphopenia, a transient reduction of blood lymphocyte counts long before the onset of clinical symptoms. We have investigated lymphopenia in mice infected with vesicular stomatitis virus (VSV) or treated with the Toll-like receptor (TLR) agonists poly(I:C) and R-848. In all cases analyzed, lymphopenia was critically dependent on type I interferon receptor (IFNAR) signaling. With the use of bone marrow–chimeric mice, radioresistant cells, such as stroma and endothelium, could be excluded as type I interferon (IFN-α/β) targets for the induction of lymphopenia. Instead, adoptive transfer experiments and studies in conditionally gene-targeted mice with a B- or T-cell–specific IFNAR deletion demonstrated that IFN-α/β exerted a direct effect on lymphocytes that was necessary and largely sufficient to induce lymphopenia. Furthermore, after treatment with R-848, we found that other cytokines such as TNF-α also played a role in T-cell lymphopenia. Investigation of the molecular mechanism revealed that lymphopenia was mainly independent of G protein–coupled receptors (GPCRs) and chemokines. In an adhesion assay, B cells of poly(I:C)–treated mice showed moderately increased adhesion to ICAM-1 but not to VCAM-1. In conclusion, our data identify a new effect of direct IFN-α/β stimulation of lymphocytes that profoundly affects lymphocyte redistribution.