Understanding the relationships among immune cells in the setting of immunologic tolerance is imperative to maintain organ and tissue transplants. T cells and natural killer (NK) cells are responsible for both immune tolerance and immune rejection; however, there is only limited knowledge about the relevance of T and NK cells in tolerance. To address this issue, we explored the possible actions of tolerant T cells on NK cells by the means of mixed lymphocyte co-cultures and NK cytotoxicity assays. We showed that tolerant T cell-induced blockade of the co-stimulatory pathway significantly inhibited NK cell function in vitro regarding antigen-presenting cells. This action was cell-cell-contact dependent. We argue that tolerant T cells and NK cells impart synergistic cooperation to maintain transplant tolerance.