The organisation of a cell’s interior, the cytoskeleton and organelles, is fundamental for every cell’s functionality. However, unlike most differentiated cells, our knowledge about the contribution of the sub-cellular architecture to embryonic development and pluripotency remains scarce. Using advanced live imaging, we discovered polarised non-centrosomal microtubules as central player for the pluripotent state of the in vivoearly mammalian embryo and induced pluripotent stem cells (iPSCs). Each cell contains an apical pole highly enriched with CAMSAP3-nucleated microtubules, growing in a longitudinal direction towards the base of PSCs. These non-centrosomal microtubules initiate an asymmetry of metabolites and organelles, including mitochondria. We further establish their rearrangement upon differentiation in a germ layer-specific manner. Dissecting how intrinsic cellular regulation contributes to pluripotency will lead a revolutionary era of regenerative and reproductive medicine.