Gammaherpesviruses establish a life-long chronic infection that is tightly controlled by the host immune response. We previously demonstrated that viruses lacking the gammaherpesvirus 68 (γHV68) viral cyclin (v-cyclin) exhibited a severe defect in reactivation from latency and persistent replication. In this analysis of chronic infection, we demonstrate that the v-cyclin is required for γHV68-associated mortality in B-cell-deficient mice. Furthermore, we identify the v-cyclin as the first gene product required for maintenance of gammaherpesvirus latency in vivo in the absence of B lymphocytes. While the v-cyclin was necessary for maintenance of latency in the absence of B cells, maintenance of v-cyclin-deficient viruses was equivalent to that of wild-type γHV68 in the presence of B cells. These results support a model in which maintenance of chronic γHV68 infection requires v-cyclin-dependent reactivation and reseeding of non-B-cell latency reservoirs in the absence of B cells and raise the possibility that B cells represent a long-lived latency reservoir maintained independently of reactivation. These results highlight distinct mechanisms for the maintenance of chronic infection in immunocompetent and B-cell-deficient mice and suggest that the different latency reservoirs have distinct gene requirements for the maintenance of latency.