This study investigates the role of histone H3 lysine 4 mono-methylation (H3K4me1) in enhancer function during embryonic stem cell (ESC) differentiation. The authors found that while KMT2C/D catalytic activities are essential for H3K4me1 and enhancer-promoter (E-P) interactions at a subset of candidate enhancers induced during neural differentiation, a majority of enhancers retain H3K4me1 in KMT2C/D catalytic mutant cells. Surprisingly, acute depletion of KMT2B, which is known to contribute to H3K4me1 at promoters, resulted in reduced H3K4me1 signals at KMT2C/D-independent enhancers and aggravated transcriptional defects. These findings suggest that KMT2B plays a crucial role in H3K4me1 deposition at enhancers, facilitating E-P interactions and enhancer-driven transcription. The study provides new insights into the mechanisms of H3K4me1 in enhancer function and its role in gene activation during differentiation.This study investigates the role of histone H3 lysine 4 mono-methylation (H3K4me1) in enhancer function during embryonic stem cell (ESC) differentiation. The authors found that while KMT2C/D catalytic activities are essential for H3K4me1 and enhancer-promoter (E-P) interactions at a subset of candidate enhancers induced during neural differentiation, a majority of enhancers retain H3K4me1 in KMT2C/D catalytic mutant cells. Surprisingly, acute depletion of KMT2B, which is known to contribute to H3K4me1 at promoters, resulted in reduced H3K4me1 signals at KMT2C/D-independent enhancers and aggravated transcriptional defects. These findings suggest that KMT2B plays a crucial role in H3K4me1 deposition at enhancers, facilitating E-P interactions and enhancer-driven transcription. The study provides new insights into the mechanisms of H3K4me1 in enhancer function and its role in gene activation during differentiation.