Myelin abnormalities, which have no effective treatment to-date, underlie numerous debilitating neurological disorders like Multiple Sclerosis (MS). While lipids constitute ~70% of myelin, the specific lipids critical for establishment of myelin sheath, and the mechanisms linking lipid metabolism to myelin assembly, have remained poorly defined, hindering therapeutic development. We generated central nervous system-specific (cKO-nestin) and oligodendrocyte-specific (cKO-OL) conditional knockout mice for Ceramide Synthase 2 (CerS2), which synthesizes very long-chain (VLC; C22–C24) ceramides. A multi-omics approach combining lipidomics, proteomics, and high-resolution mass spectrometry imaging (MSI) was employed to delineate the lipid and protein aberrations upon CerS2 knockout. Functional consequences were assessed using behavioral tests, electron microscopy, and in vitro myelination assays of dorsal root ganglion (DRG) neuron-oligodendrocyte precursor cells (OPCs) co-cultures. Our findings define a novel pathogenic cascade where cell-autonomous loss of CerS2 impedes oligodendrocyte differentiation and depletes VLC sphingolipids, destabilizing membrane microdomains and impairing MBP localization, culminating in myelination failure. This work positions the CerS2-VLC sphingolipid axis as a potential therapeutic target for demyelinating diseases.