Lipid rafts are packed nanoscopic domains on plasma membrane and essential signalling platforms for transducing extracellular stimuli into cellular responses. Although depletion of raft component glycoshpingolipids causes abnormality particularly in ectoderm layer formation, it remains unclear whether rafts play a role in lineage determination, a critical but less-known stage in lineage commitment. Here, inducing mouse embryonic stem cell (mESC) differentiation with retinoic acid (RA), we observed lipid rafts increased since early stage, especially in ectoderm-like cells. Stochastic optical reconstruction microscopy characterized at super-resolution the distinct raft features in mESCs and the derived differentiated cells. Furthermore, RA-induced commitment of ectoderm-like cells was significantly diminished not only by genetic ablation of rafts but by applying inhibitor for glycosphingolipids or cholesterol at early differentiation stages. Meanwhile, raft inhibition delayed RA-induced pluripotency exit, an early step required for differentiation. Therefore, lipid rafts increase and facilitate ectoderm lineage specification as well as pluripotency exit during mESC differentiation.