<p>As a crucial lymphocyte subset, γδT cells exhibit tumoricidal properties without the constraint of major histocompatibility complex restriction. In recent years, the in vitro expansion of human peripheral blood-derived γδT cells has emerged as a promising candidate for adoptive immune cell therapy (ACT). However, given the limitations in accessing and sourcing human peripheral blood, it remains challenging to expand γδT cells to the magnitude required for clinical cancer immunotherapy. Therefore, the objective of this study was to establish an innovative approach for the in vitro differentiation of γδT cells from human embryonic stem cells (hESCs). In present study, by simulating the in <i>vivo</i> developmental process of γδT cells, we established stroma cell-free differentiation approach with three stages under 3D suspension culture in conjunction with using hypoxic conditions. Under our protocol, around 39% of CD34⁺CD31⁺ hematopoietic endothelial cells were induced in cell spheroids at first stage under hypoxic conditions, and over 99% of suspended/floating cells from cell spheroids were early CD43⁺ hematopoietic progenitor cells (HPCs), and over 90% of CD45⁺ HPCs were produced at the second stage, demonstrating the homogeneity of cell population and its high hematopoietic potential. Eventually over 40% of mature CD45⁺CD3⁺TCRγδ⁺ T cells were generated at the final stage, and these γδT cells exerted cytotoxicity against hepatoma cancer cells and simultaneously significantly inhibited their proliferation. RNA sequencing analysis revealed that the differentiation process of hESC-derived γδT cells was transcriptomically similar to those of in vivo courses. Therefore, our findings ultimately offer a novel technique to address the challenge of sourcing γδT cells for ACT therapies.</p>