Generation of T lymphocytes with reactivity against cancer is a prerequisite for effective adoptive cellular therapies. We established a protocol for tumor-infiltrating lymphocytes (TILs) from patients with pancreatic ductal adenocarcinoma. Tumor samples from 17 pancreatic cancer specimens were cultured with cytokines (IL-2, IL-15, and IL-21) to expand TILs. After 10 days of culture, TILs were stimulated with an anti-CD3 antibody (OKT3) and irradiated allogeneic peripheral blood mononuclear cells. Reactivity of TILs against tumor-associated antigens (mesothelin, survivin, or NY-ESO-1) was detected by intracellular cytokine production by flow cytometry. Cytotoxicity was measured using a Chromium 51 release assay, and reactivity of TILs against autologous tumor cells was detected by INF-γ production (ELISA). TIL composition was tested by CD45RA, CCR7, 4-1BB, LAG-3, PD-1, TIM3, and CTLA-4 marker analysis. TCR Vβ was determined by flow cytometry and TCR clonality was gauged measuring the CDR3 region length by PCR analysis and subsequent sequencing. We could reliably obtain TILs from 17/17 patients with a majority of CD8+ T cells. CD3+CD8+, CD3+CD4+, and CD3+CD4−CD8− [double-negative (DN) T cells] resided predominantly in central (CD45RA−CCR7+) and effector (CD45RA−CCR7−) memory subsets. CD8+ TILs tested uniformly positive for LAG-3 (about 100%), whereas CD4+ TILs showed only up to 12% LAG-3+ staining and PD-1 showed a broad expression pattern in TILs from different patients. TILs from individual patients recognized strongly (up to 11.9% and 8.2% in CD8+) NY-ESO-1, determined by ICS, or mesothelin, determined respectively by TNF-α and IFN-γ production. Twelve of 17 of CD8+ TILs showed preferential expansion of certain TCR Vβ families (eg, 99.2% Vβ13.2 in CD8+ TILs, 77% in the Vβ1, 65.9% in the Vβ22, and 63.3% in the Vβ14 family). TCR CDR3 analysis exhibited monoclonal or oligoclonal TCRs, some of them (eg, CD8+ Vβ13.2) reacting strongly against autologous tumor defined by INF-γ production or by cytotoxicity. We have optimized methods for generating pancreatic cancer–specific TILs that can be used for adoptive cellular therapy of patients with pancreatic cancer.