<h3>Background</h3> Adoptive cell transfer of tumor-infiltrating lymphocytes (TILs) has emerged as a prominent therapeutic strategy involving the isolation, in vitro expansion, and reinfusion of TILs derived from resected tumors. Traditionally, TIL isolation has relied on high concentrations of IL-2. However, recent research explores incorporating CD3 (OKT3) and 4-1BB (Urelumab) alongside IL-2 during initial TIL isolation to activate all three lymphocyte signals. This approach has demonstrated reduced culture times and increased CD8+ cell percentages in melanoma and lung cancer. Nevertheless, the phenotypic effects of these stimuli on TILs remain largely unexplored. In this study, we evaluate TILs derived from melanoma tumors stimulated with IL-2 + OKT3, either alone or in combination with Urelumab. <h3>Methods</h3> Eight melanoma tumors from Colombia's National Cancer Institute patients were cultured with or without Urelumab. Post-expansion, TILs were assessed using flow cytometry with a multicolor panel for memory, activation, and exhaustion markers. Manual and automated co-expression analysis of flow cytometry data and RNA sequencing were conducted to evaluate differential phenotype and expression. <h3>Results</h3> Employing all three T cell activation signals from tumor fragments accelerates TIL growth, averaging 15.75 days versus 25 days without Urelumab. Flow cytometry revealed comparable CD3 (&gt;90%), CD4 (&lt;10%), CD8 (&gt;85%) percentages, and memory phenotype (CD45RA-CCR7-), and intracellular cytokine expression (IFN-γ, TNF-α, IL-2) following polyclonal stimulation. However, 4-1BB agonist inclusion showed trends towards tumor-reactive populations (PD-1, CD103, CD39) and higher CD69-CD39- populations observed in responsive melanoma TIL therapy. A preliminary analysis of differential gene expression indicates a downmodulation of CD3 signaling in cells cultured with Urelumab. <h3>Conclusions</h3> These findings suggest that co-stimulatory signals during the pre-REP phase enhance TIL growth rates without altering phenotype, promoting markers associated with tumor-reactive cells. Further analysis of gene expression is necessary to confirm this finding and explore its implication in t cell biology. <h3>Ethics Approval</h3> The study was approved by the Ethics Commitee of Instituto Nacional de Cancerología (Bogota, Colombia) on October 28th, 2020. Name of the study:'Analisis del perfil inmunologico de muestras de tumores metastasicos en pacientes con melanoma localmente avanzado y metastasico del Instituto Nacional de Cancerologia'. ID of the aproval: CEI-0026-20. The participans gave informed consent before taking part of the study.