CD8+ T cell anergy is a critical mechanism of peripheral tolerance, poorly investigated in response to immunotherapy. Here, using a pancreatic islet allograft model and CD3 antibody therapy, we showed, by single cell gene profiling, that intragraft CD8+ lymphocytes coexpressing granzyme B and perforin were selectively depleted through the Fas/FasL pathway. This step led to long-standing anergy of the remaining CD8+ T cells marked by the absence of cytotoxic/inflammatory gene expression also confirmed by transcriptome analysis. This sustained unresponsiveness required the presence of the alloantigens. Furthermore, tissue-resident CD8+ lymphocytes produced TGFβ and expressed the inhibitory receptors PD-1 and PD-L1. Blockade of TGFβ downregulated PD-1 and PD-L1 expression and precipitated graft rejection. Neutralizing PD-1, PD-L1 or TGFβRII signaling in T cells also abrogated CD3 antibody-induced tolerance. These studies unravel novel mechanisms underlying CD8+ T cell anergy and reveal a cell intrinsic regulatory link between the TGFβ and the PD-1/PD-L1 pathways.