Generation of tumor-antigen specific CD4+ T-helper (T H) lines through in vitro priming is of interest for adoptive cell therapy of cancer, but the development of this approach has been limited by the lack of appropriate tools to identify and isolate low frequency tumor antigen-specific CD4+ T cells. Here, we have used recently developed MHC class II/peptide tetramers incorporating an immunodominant peptide from NY-ESO-1 (ESO), a tumor antigen frequently expressed in different human solid and hematologic cancers, to implement an in vitro priming platform allowing the generation of ESO-specific T H lines. We isolated phenotypically defined CD4+ T-cell subpopulations from circulating lymphocytes of DR52b+ healthy donors by flow cytometry cell sorting and stimulated them in vitro with peptide ESO 119-143, autologous APC and IL-2. We assessed the frequency of ESO-specific cells in the cultures by staining with DR52b/ESO 119-143 tetramers (ESO-tetramers) and TCR repertoire of ESO-tetramer+ cells by co-staining with TCR variable β chain (BV) specific antibodies. We isolated ESO-tetramer+ cells by flow cytometry cell sorting and expanded them with PHA, APC and IL-2 to generate ESO-specific T H lines. We characterized the lines for antigen recognition, by stimulation with ESO peptide or recombinant protein, cytokine production, by intracellular staining using specific antibodies, and alloreactivity, by stimulation with allo-APC. Using this approach, we could consistently generate ESO-tetramer+ T H lines from conventional CD4+ CD25− naïve and central memory populations, but not from effector memory populations or CD4+ CD25+ Treg. In vitro primed T H lines recognized ESO with affinities comparable to ESO-tetramer+ cells from patients immunized with an ESO vaccine and used a similar TCR repertoire. In this study, using MHC class II/ESO tetramers, we have implemented an in vitro priming platform allowing the generation of ESO-monospecific polyclonal T H lines from non-immune individuals. This is an approach that is of potential interest for adoptive cell therapy of patients bearing ESO-expressing cancers.