Lysine-specific histone demethylase (KDM) 5 inhibition by KDM5-C70 induces astrocytogenesis and highlights the importance of modulation of histone methylation in cell fate specification. This study investigated the role of the histone demethylase inhibitor KDM5-C70 in modulating the metabolic and lipidomic landscape during astrocyte differentiation of rat neural stem cells (NSCs). Using chemical derivatisation combined with gas chromatography-mass spectrometry, 42 metabolites were detected, indicating potential regulation of phospholipid metabolism. Subsequent lipidomic analysis, employing reverse-phase liquid chromatography with high-resolution quadrupole time-of-flight mass spectrometry, identified 180 lipid species and 9 lipid subclasses. Integrative analysis revealed that KDM5-C70 promoted astrocytogenesis through epigenetic changes linked to the attenuation of phosphatidylethanolamine (PE) biosynthesis pathways. The reduced expression of transcripts related to PE highlighted the significance of the PE pathway in influencing cell fate decisions. These quantitative metabolomic and lipidomic analyses not only advance our understanding of NSC differentiation but also lay the groundwork for potential therapeutic strategies targeting metabolic pathways in neurodegenerative diseases and neural injuries.