Background: A promising avenue to increase the efficacy of anti-CTLA-4 and anti-PD-(L)1 checkpoint blockade therapies is to enhance T cell induction through vaccination. Surprisingly, concurrent gp100 peptide vaccination decreased clinical efficacy of anti-CTLA-4 in patients with melanoma. As a result, it is currently unclear how to effectively combine checkpoint blockade therapy with vaccination. We used an animal model of gp100 peptide vaccination and CTLA-4/PD-L1 checkpoint blockade to understand this phenomenon and define parameters for successful combination of checkpoint blockade and vaccination. Methods: We treated established B16 melanoma tumors with anti-CTLA-4 therapy and concurrent vaccination with gp100/IFA and studied T cell expansion, trafficking and therapeutic effector function. Results: Anti-CTLA-4 monotherapy significantly increased intratumoral levels of TRP-2 melanoma antigen-specific Teffs, as well as total CD44^hiCD11a^hiCD8^lo Teffs at the tumor site. Conversely, gp100/IFA vaccination-induced, gp100-specific CD8+ T cells accumulated at the inflamed vaccination site. Surprisingly, combination of gp100/IFA vaccination and anti-CTLA-4 therapy caused non-gp100-specific Teff, induced by anti-CTLA-4 therapy, to also accumulate at the vaccination site and not at the tumor site. This Teff sequestration at the vaccination site was dependent on IFN-g, CXCR3 and ICAM-1. Replacing the gp100/IFA formulation with a gp100-encoding viral vector or a non-persistent, water-based formulation potently synergized with anti-CTLA-4 or anti-PD-L1 resulting in a high proportion of complete cure. Conclusions: Persistent vaccine formulations can fail to synergize, or even diminish the efficacy of, CTLA-4/PD-(L)1 checkpoint–based cancer immune therapy through the induction of aberrant T cell trafficking to the vaccination site. A non-persistent vaccine formulation can reverse these undesirable effects and potently synergize with anti-CTLA-4 and/or anti-PD-(L)1 checkpoint blockade, resulting in significantly improved anti-tumor activity.