During neuronal differentiation, the transcriptional profile and the epigenetic context of neural committed cells is subject to significant rearrangements, but a systematic quantification of global histone modification changes is still missing. Here, we show that H3K79me2 increases and H3K27ac decreases globally during in-vitro neuronal differentiation of murine embryonic stem cells. DOT1L mediates all three degrees of methylation of H3K79 and its enzymatic activity is critical to modulate cellular differentiation and reprogramming. In this context, we find that inhibition of DOT1L in neural progenitor cells biases the transcriptional state towards neuronal differentiation, resulting in transcriptional upregulation of genes marked with H3K27me3 on the promoter region. We further show that DOT1L inhibition affects accessibility of SOX2-bound enhancers and impairs SOX2 binding in neural progenitors. Our work provides evidence that DOT1L activity gates differentiation of progenitors by allowing SOX2-dependent transcription of stemness programs.