Immunotherapy, especially the adoptive transfer of T cells and immune checkpoint blockade therapy, have revolutionized cancer therapy. In particular, utilizing antigen-specific T cells for adoptive cell therapy has enabled the development of specific and effective strategies. It has paved the way for developing more accurate and personalized cancer immunotherapies. Adoptive cell therapy (ACT) results depend on the characteristics of ex vivo expanded T cells, such as their differentiation and clonal diversity. However, ex vivo expanded specific T cells often express several inhibitory receptors involved in T-cell exhaustion and markers of terminal effector differentiation. Accordingly, we hypothesized that blocking one or several inhibitory receptors during the ex vivo expansion could improve the expansion and differentiation of antigen-specific T cells. Preconditioning the ACT products and combinatorial immunotherapy approaches are newly developed concepts in cancer therapy to optimize cancer immunotherapy for a larger group of patients. To study the development of antigen-specific T-cells in combination with checkpoint blockade, we have adopted a method that allows the expansion of rare antigen-specific T cell precursors from PBMCs via multiple stimulations, using antigen-pulsed dendritic cells. In the current study, we utilized our protocol to generate and expand antigen-specific CD8+ T cells targeting the oncogenic Epstein-Barr virus (EBV)-LMP2 and a tumor-associated antigen (TAA) from the Wilms Tumor 1 (WT1) protein. We employed two approaches to abolish the negative regulatory receptors, antibody-mediated blockade and deletion via CRISPR/Cas9. We evaluated the impact of checkpoint blockade on antigen-specific T cells development, proliferation, and function. Additionally, TCR clonality and transcriptomic changes were assessed by genomic studies, including single-cell RNA (scRNA) sequencing and T-cell receptor sequencing. Supporting our hypothesis, we observed that blocking both PD-L1 and TIM3 (not any of them alone) significantly enhanced LMP2 and WT1-specific T cell generation and expansion. Additionally, checkpoint blockade resulted in higher specific T cell function, including cytokine production and in vitro targeted cytotoxicity. Using scRNA-seq and TCR sequencing approaches, we first remarked that the specific T cells are highly oligoclonal and identified a few dominant shared clones between donors. Immune checkpoint blockade did not confer consistent transcriptional signatures but may have a clonotype and donor-specific impact on the expression of activation and exhaustion-related genes. Overall, immune checkpoint blockade did not markedly alter the clonal composition of the T-cell product. We also evaluated the impact of CD5 deletion in antigen-specific T cell priming and expansion as an inhibitory receptor and a part of the immune response synapse. However, in a human ACT setting, our data show that the CRISPR/Cas9 mediated CD5 deletion only has modest effects on antigen-specific T-cell generation. However, future combinations with the blockade of other immune checkpoint may be warranted. Conclusion We demonstrated that blocking PD-L1 and TIM3 during the ex vivo expansion improves antigen-specific T-cell yield. We show that blocking multiple checkpoints can synergistically optimize specific T-cell production without compromising the response's specificity. It is a rapidly implementable strategy to enhance the number and quality of ex vivo expanded antigen-specific T cells for immunotherapy.