In the central nervous system, Neurogenin 3 (Ngn3) is a proneural transcription factor that regulates neuritogenesis and its expression is regulated by a sex-specific enrichment of H3K27m3 in hypothalamic neurons from male embryos. Previous evidence from our laboratory demonstrated that the histone methylase Ezh2, is sexually dimorphic in hypothalamic tissue suggesting that its higher expression in male neurons may regulate Ngn3. In this study, we evaluate the sex-specific expression and regulation of Ezh2 in ventromedial hypothalamic neurons in vitro. To this purpose, we performed primary cultures from male and female mouse embryos at embryonic age 15 (E15). On day 3 in vitro, we evaluated the basal and 17β-estradiol (E2, 10-10M) induced mRNA expression of Ezh2. Furthermore, we pharmacologically inhibited EZH2 using UNC1999 (2µM) and quantified Ngn3 mRNA expression levels by qPCR. Results indicate no sex differences in Ezh2 expression (p=0.14; n=5-6) and no effect of E2 (p=0.44; n=4-5). However, pharmacological inhibition of EZH2 significantly upregulates Ngn3 gene expression only in female neurons (p=0.05; n=6). These findings suggest that the H3K27m3 methylation by EZH2 might regulate hypothalamic neuronal differentiation in a sex-specific manner. Thus, the observed sex differences could result from a balance of activating/repressing epigenetics marks on the Ngn3 promoter, which in turn may depend on the key role of the histone demethylase KDM6A.