Wilms’ tumor protein 1 (WT1) is a tumor-associated antigen overexpressed in various cancers. As a self-antigen, negative selection reduces the number of WT1-specific T cell receptors (TCRs). Here, we provide a protocol to generate WT137-45-specific TCRs using healthy human peripheral blood mononuclear cells. We describe the expansion of WT1-specific T cell clones by two consecutive in vitro stimulations with autologous WT137-45-pulsed dendritic cells and peripheral blood lymphocytes. We then detail the detection with human leukocyte antigen/WT137-45 tetramers. Display omitted •Robust fully autologous in vitro expansion of WT1-reactive T cells from healthy donors•Two consecutive one-week stimulations with autologous peptide-pulsed DCs and PBLs•Steps for tetramer staining, allowing WT137-45-specific T cell purification•Expanded T cells can serve for applications analyzing TCR repertoire or isolating TCRs Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Wilms’ tumor protein 1 (WT1) is a tumor-associated antigen (TAA) overexpressed in various cancers. As a self-antigen, negative selection reduces the number of WT1-specific T cell receptors (TCRs). Here, we provide a protocol to generate WT137-45-specific TCRs using healthy human peripheral blood mononuclear cells. We describe the expansion of WT1-specific T cell clones by two consecutive in vitro stimulations with autologous WT137-45-pulsed dendritic cells and peripheral blood lymphocytes. We then detail the detection with human leukocyte antigen/WT137-45 tetramers.