The epigenetic status, which regulates the cellular identity and differentiation potential of pluripotent stem (PS) cells, dynamically responds to the culture environment, affecting the safe and effective use of PS cells for basic research and therapeutic applications. However, the key mediator(s) representing the epigenetic signatures of PS cells under distinct culture conditions remains unclear. Here we investigated the role of DNA methyltransferase 3-like (Dnmt3L) in modulation of the DNA methylation and differentiation potential of mouse embryonic stem (ES) cells. Unlike other de novo DNA methyltransferases, Dnmt3L exhibited a uniquely dynamic expression pattern during prolonged 2i-leukemia inhibitory factor culture, which was marked by rapid post-transcriptional upregulation that sensitively reflected changes in the extracellular environment. Mass spectrometry identified that acetylation of lysine residues K238 and K412 controlled Dnmt3L protein stability. This site-specific acetylation critically modulated expression of genes associated with naive pluripotency and lineage differentiation-especially toward germline, neural and cardiac fates-through targeted DNA methylation and thereby orchestrated the lineage-specific developmental potential of mouse ES cells both in vitro and in vivo. Our findings demonstrate that Dnmt3L is a key regulator of epigenetic stability at developmentally critical loci in mouse ES cells and dynamically responds to changes in the extracellular culture environment. Thus, elucidation of the regulatory mechanism of Dnmt3L may provide insight into the onset of epigenetic aberrations and suggest the optimal culture conditions to preserve the epigenetic integrity of ES cells, which has significant implications for regenerative medicine.