T cell receptor (TCR) signalling is crucial in determining the fate of thymocyte differentiation in the thymus. The high-avidity interaction between TCR and self-peptide-MHC complexes induces development of regulatory T cells (Tregs), lineage commitment for which is controlled by expression of transcription factor Forkhead box P3 (FoxP3). The non-catalytic region of the tyrosine kinase (Nck) comprises two members, Nck1 and Nck2, with Nck1 playing a dominant role in TCR-mediated T cell activation and function. Nck's role, while established in thymocyte development, remains unelucidated in development of Tregs. In this study, we aimed to determine the function of Nck1 in the in vitro development and differentiation of human thymocytes. Human thymocytes were transfected with shRNA plasmid to silence Nck1 expression. The number of FoxP3+ Tregs decreased noticeably in Nck1 knockdown thymocytes after co-cultivation with myeloid dendritic cells (mDCs) and thymic epithelial cells for 14 days. Furthermore, decreased phosphorylation of AKT and FoxO1 was observed in Nck1-silenced thymocytes, in association with reduced FoxO1 nuclear localization. Taken together, these findings identify the pivotal role of Nck1 in Treg development.