Maximizing the potential of cancer immunotherapy requires model systems that closely recapitulate human disease to study T-cell responses to tumor antigens and to test immunotherapeutic strategies. We have created a new system that is compatible with Cre-LoxP–regulatable mouse cancer models in which the SIY antigen is specifically overexpressed in tumors, mimicking clinically relevant TAAs. To show the utility of this system, we have characterized SIY-reactive T cells in the context of lung adenocarcinoma, revealing multiple levels of antigen-specific T-cell tolerance that serve to limit an effective antitumor response. Thymic deletion reduced the number of SIY-reactive T cells present in the animals. When potentially self-reactive T cells in the periphery were activated, they were efficiently eliminated. Inhibition of apoptosis resulted in more persistent self-reactive T cells, but these cells became anergic to antigen stimulation. Finally, in the presence of tumors overexpressing SIY, SIY-specific T cells required a higher level of costimulation to achieve functional activation. This system represents a valuable tool in which to explore sources contributing to T-cell tolerance of cancer and to test therapies aimed at overcoming this tolerance. [Cancer Res 2008;68(22):9459–68]