A pro-oligodendroblast-specific monoclonal antiboby O4 antigen named pro-oligodendroblast antigen (POA) has not been identified biochemically, whereas it has been widely used as a useful marker for this critical stage in the oligodendrocyte lineage. The O4 antibody reacts to sulfatide (HSO3-3-galactosylceramide) at the mature oligodendrocyte stage, but sulfatide synthesis at the pro-oligodendroblast stage is uncertain. In the present study we show by using imaging mass spectrometry that sulfatide exists at restricted regions of the ventricular zone of spinal cord in both of the stage 32 chick embryo and of the embryonic day 13 mouse fetus, where pro-oligodendroblasts first appear. There are various sulfatide isoforms with different fatty acid composition. We found that short-chain sulfatide with 18 and 20 carbon atoms (C18 and C20, respectively) are dominant during the initial appearance of pro-oligodendroblasts in the embryonic spinal cord, while long-chain (C24) sulfatide is dominant in adult spinal cord. However, in a later stage of oligodendrocyte maturation at mouse embryonic day 19, sulfatides with C22 or C24 become to be dominant. These results indicated that pro-oligodendroblasts have short-chain sulfatides and their fatty acid chain length changes with oligodendeocyte maturation stage and age. Moreover, O4-positive and O1 (anti-GalCer)-negative pro-oligodendroblasts were sorted using a flow cytometer and then their sulfatide forms were determined by LCMS-IT-TOF. The data show that the pro-oligodendroblasts have C18 sulfatide. Together, these results elucidate that POA at pro-oligodendroblast stage is the sulfatide with short chain fatty acids.