New approaches based on induction of antigen-specific immunological tolerance are being explored for treatment of autoimmunity and prevention of immunity to protein drugs. Antigens associated with apoptotic debris are known to be processed tolerogenically in vivo. Our group is exploring an approach toward antigen-specific tolerization using erythrocyte-binding antigens, based on the premise that as the erythrocytes circulate, age and are cleared, the erythrocyte surface-bound antigen payload will be cleared tolerogenically along with the eryptotic debris. Here, we characterized the phenotypic signatures of CD8+ T cells undergoing tolerance in response to soluble and erythrocyte-targeted antigen. Signaling through programmed death-1/programmed death ligand-1 (PD-1/PD-L1), but not through cytotoxic T lymphocyte antigen 4 (CTLA4), was shown to be required for antigen-specific T cell deletion, anergy and expression of regulatory markers. Generation of CD25+FOXP3+ regulatory T cells in response to erythrocyte-targeted antigens but not soluble antigen at an equimolar dose was observed, and these cells were required for long-term maintenance of immune tolerance in both the CD4+ and CD8+ T cell compartments. Evidence of infectious tolerance was observed, in that tolerance to a one antigenic epitope was able to regulate responses to other epitopes in the same protein antigen.