The use of autologous T cells expressing chimeric antigen receptors (CAR) has revolutionized the treatment of hematological malignancies. However, the use of allogeneic CAR-T cells offers many advantages over autologous therapies, including the immediate availability of cryopreserved batches for infusion and the use of healthy donor cells since it is possible to obtain high amounts of fully functional cells. Moreover, this strategy also allows for the scaling up of the production of CAR-T cells resulting in cost reduction. Cells that do not express TCR αβ, such as gamma delta (γδ) T, are an alternative source to allow widespread allogeneic therapy possible. Their ability to recognize targets in an MHC-independent manner and the expression of innate immunity receptors increases the potential of using these cells in immunotherapies. Aiming to improve the effectiveness of γδ T-cell-based immunotherapies against tumors, we tested various optimization strategies. The most widely used protocol for selectively expanding Vδ2T cells relies on zoledronic acid (ZOL) stimulation of PBMCs in combination with IL-2 or/and IL-15 cytokines. Here, we tested different concentrations of IL-2 (100 and 1,000 U/mL) and IL-15 (12.5 and 100 ng/mL) with 5 μM zoledronic acid. In addition, we compared cell expansion in three defined cell culture media, OpTmizer T Cell Expansion SFM, X-vivo10, and TexMACS (supplemented with 2% AB serum). A total of twenty-four different combinations of IL-2, IL-15, and media were tested in the present study.After twelve days of culture, cells were counted and labelled with anti-CD3, anti-CD8, anti-CD4, anti-Vδ2, anti-PD1, anti-CD69, and anti-NKG2D antibodies for flow cytometry analysis. Our results demonstrated that although TexMACS medium is capable of supporting T-cell activation and expansion, it was not optimal for maximal expansion of Vδ2T cells (fold expansion 5.99 ± 1.61). However, the fold expansion was similar in OpTmizer and X-vivo10 media (fold expansion 21.38 ± 5.13 and 18.37 ± 5.42, respectively). Among the medium supplementation strategies, cultivation in OpTmizer combined with 100 U/mL IL-2 + 12.5 ng/mL IL-15 led to highest expansion index (32.4-fold). As for the activation markers of Vδ2T cells, the frequency of NKG2D+ cells was similar for all conditions tested (above 85%). However, the percentage of CD69+ cells was higher in cells cultured in TexMACS medium compared to OpTmizer and X-vivo10 media (81.6±5.3, 69±9.9, 60.2±7.2, respectively). It is known that the Programmed Cell Death-1 receptor (PD-1) plays an important role in regulating immune cell function. We examined the expression of PD-1 after twelve days of expansion. We observed lower levels of PD-1 in cells expanded in X-vivo10 medium supplemented with 1,000 U/mL IL-2 + 100 ng/mL IL-15, whereas levels of expression were up-regulated significantly when cells were cultivated in the same conditions without IL-15 (28.7% versus 70.6%). Cells expanded in either OpTimizer or TexMACS demonstrated a similar frequency to PD-1 positive cells (41±13% and 49.7%±9.5). In conclusion, we reported an optimized protocol to improve the expansion and the quality of Vδ2 T cells. These findings set the groundwork for the establishment of an off-the-shelf advanced cellular immunotherapy using this alternative population of T cells.