Delivery of gene therapy as well as of biologic therapeutics is often hampered by the immune response of the subject receiving the therapy. We have reported that effective gene therapy for hemophilia utilizing platelets as a delivery vehicle engenders profound tolerance to the therapeutic product. In this study, we investigated whether this strategy can be applied to induce immune tolerance to a non-coagulant protein and explored the fundamental mechanism of immune tolerance induced by platelet-targeted gene delivery. We used ovalbumin (OVA) as a surrogate non-coagulant protein and constructed a lentiviral vector in which OVA is driven by the platelet-specific αIIb promoter. Platelet-specific OVA expression was introduced by bone marrow transduction and transplantation. Greater than 95% of OVA was stored in platelet α-granules. Control mice immunized with OVA generated OVA-specific IgG antibodies; however, mice expressing OVA in platelets did not. Furthermore, OVA expression in platelets was sufficient to prevent the rejection of skin grafts from CAG-OVA mice, demonstrating that immune tolerance developed in platelet-specific OVA-transduced recipients. To assess the mechanism(s) involved in this tolerance we used OTII mice that express CD4+ effector T cells specific for an OVA-derived peptide. After platelet-specific OVA gene transfer, these mice showed normal thymic maturation of the T cells ruling against central tolerance. In the periphery, tolerance involved elimination of OVA-specific CD4+ effector T cells by apoptosis and expansion of an OVA-specific regulatory T cell population. These experiments reveal the existence of natural peripheral tolerance processes to platelet granule contents which can be co-opted to deliver therapeutically important products.