Natural killer (NK) cells are essential in controlling cancer and infection. However, little is known about the dynamics of the transcriptional regulatory machinery during NK cell differen-tiation. In this study, we applied the assay of transposase accessible chromatin with sequencing (ATAC-seq) technique in a home-developed in vitro NK cell differentiation system. Analysis of ATAC-seq data illustrated two distinct transcription factor (TF) clusters that dynamically regulate NK cell differentiation. Moreover, two TFs from the second cluster, FOS-like 2 (FOSL2) and early growth response 2 (EGR2), were identified as novel essential TFs that control NK cell maturation and function. Knocking down either of these two TFs significantly impacted NK cell differentia-tion. Finally, we constructed a genome-wide transcriptional regulatory network that provides a bet-ter understanding of the regulatory dynamics during NK cell differentiation.