Human cord blood–derived γδ T cells (CBγδ) display a highly diverse TCRγδrepertoire and have a unique subtype composition different from fetal or adult peripheral blood counterparts. We expanded CBγδin vitro using an irradiated Epstein-Barr virus–transformed feeder cell–based modified rapid expansion protocol (REP). Single-cell RNA sequencing tracked progressive differentiation of naïve CBγδinto cells expressing neoantigen-reactive tumor-infiltrating lymphocyte as well as tissue-resident memory precursor–like and antigen-presenting cell–like gene signatures. TCRγδclonal tracing revealed a bias toward cytotoxic effector differentiation in a much larger proportion of Vδ2−clones compared to Vδ2+clones, resulting in the former being more cytotoxic at the population level. These clonotype-specific differentiation dynamics were not restricted to REP and were recapitulated upon secondary nonviral antigen stimulations. Thus, our data showed intrinsic cellular differences between major subtypes of human γδ T cells already in operation at early postnatal stage and highlighted key areas of consideration in optimizing cell manufacturing processes.