Significance The emergence of the cell-autonomous circadian oscillator is coupled with cellular differentiation. Cellular differentiation, as well as reprogramming, results in global alterations of the transcriptional program via epigenetic modification such as DNA methylation. We here demonstrate that c-Myc constitutive expression and Dnmt1 ablation disrupt the differentiation-coupled emergence of the clock from mouse ES cells (ESCs). Using these model ESCs, 484 genes were identified by global gene expression analysis as factors correlated with circadian clock development. Among them, we find that misregulation of Kpna2 ( Importin-α2 ) during the differentiation culture of ESCs significantly impairs clock development, and KPNA2 facilitates cytoplasmic localization of PER1/2. These results suggest that the programmed gene expression network regulates the differentiation-coupled circadian clock development in mammalian cells.