Vaccine-induced CD8(+) T-cell responses can eradicate developing tumors in vivo in mouse models. Translating these successes into approved treatments for cancer patients has been challenging, since many of these models lack expression of clinically proven/relevant tumor antigens. We have shown that mesothelin is a clinically relevant CD8(+) T-cell target in human pancreas cancer, which is also highly conserved among species. Here, we utilize the murine mesothelin-expressing pancreatic tumor model (Panc02) to identify the immune-relevant mesothelin-derived peptides and study interventions that enhance the antitumor response. We first screened overlapping peptides of the entire murine mesothelin protein to identify two new CD8(+) mesothelin-restricted epitopes. These peptides were then evaluated for recognition by vaccine-induced T cells from mice treated with vaccine in sequence with low-dose cyclophosphamide (CY) and an anti-CD25 IL-2Ralpha monoclonal antibody (PC61). These treatments are both known to deplete subpopulations of T regulatory cells (Tregs). Our findings demonstrate that combined Treg-depleting therapies synergize to enhance vaccine efficacy. Furthermore, our data supports mesothelin as a relevant antigen in murine and clinical models and the use of Panc02 as a clinically relevant murine model of pancreatic cancer for evaluating antigen-targeted immunotherapies in immune-tolerant hosts.