Centre for Translational Stem Cell Biology 1 article published in JoVE Developmental Biology Generation of Natural Killer Cells from Human Expanded Potential Stem Cells Poon Yu Ching1, Clement Wang2, Su Hang2, Pentao Liu2,3, Ryohichi Sugimura2,3 1Major in Molecular Biology and Biotechnology, School of Biological Sciences, The University of Hong Kong, 2School of Biomedical Sciences, The University of Hong Kong, 3Centre for Translational Stem Cell Biology The present protocol shows how to differentiate CD3−/CD45+CD56+ cells with mild cytotoxicity from human expanded potential stem cells (hEPSCs) under both 3D and 2D culture conditions. This allows for routine phenotypical validation without the destruction of the complex microenvironment.