Abstract 4-1BB signals are considered positive regulators of T cell responses against viruses and tumors, but recent studies suggest that they have more complex roles in modulating T cell responses. Although dual roles of 4-1BB signaling in T cell responses have been suggested, the underlying mechanisms are still not fully understood. In this study, we tested whether 4-1BB expression affected T cell responses differently when expressed in myeloid versus lymphoid cells in vivo. By assessing the proliferation of 4-1BB+/+ and 4-1BB−/− T cells in lymphocyte-deficient RAG2−/− and RAG2−/−4-1BB−/− mice, we were able to compare the effects on T cell responses of 4-1BB expression on myeloid versus T cells. Surprisingly, adoptively transferred T cells were more responsive in tumor-bearing RAG2−/−4-1BB−/− mice than in RAG2−/− mice, and this enhanced T cell proliferation was further enhanced if the T cells were 4-1BB deficient. Dendritic cells (DCs) rather than NK or tissue cells were the myeloid lineage cells primarily responsible for the enhanced T cell proliferation. However, individual 4-1BB−/− DCs were less effective in T cell priming in vivo than 4-1BB+/+ DCs; instead, more DCs in the secondary lymphoid organs of RAG2−/−4-1BB−/− mice appeared to induce the enhanced T cell proliferation by producing and transpresenting more IL-15. Therefore, we conclude that in vivo 4-1BB signaling of myeloid cells negatively regulates peripheral T cell responses by limiting the differentiation of DCs and their accumulation in secondary lymphoid organs.