The thymus is the primary organ for T cell differentiation and maturation. Its stroma forms a characteristic sponge-like 3D structure mainly composed of thymic epithelial cells. Despite of this unconventional epithelial architecture, TECs express markers associated with epidermal specification and differentiation. We have uncovered that the human thymus contains a population of clonogenic TECs that can be extensively expanded in a culture system originally developed for skin keratinocyte stem cells. In vitro, human TECs (hTECs) can give rise to four morphologically distinct colony-types and express markers of stratified epithelia’s basal layers, such as P63, K5/K14 and CD49f. We were able to demonstrate that cultured hTECs can be split in two distinct subpopulations based on their EpCAM expression level. EpCAM+ hTECs only give rise to stratified colonies that contain squame-like cells and express markers of epidermal differentiation, whereas EpCAM- hTECs mostly give rise to non-stratifying colonies but have the capacity to generate EpCAM+ hTECs. EpCAM- hTECs maintain a basal epithelial identity but display hallmarks of EMT, such as the upregulation of ZEB1, the loss of CDH1 and a reduced expression of the miR-200 family members. We were also able to show that miR-200c overexpression is sufficient to convert EPCAM- hTECs into EpCAM+ ones, implying a crucial role for the ZEB/miR-200 double-negative feedback loop in the control of stratification and EMT in cultured hTECs. Our work suggests that hTECs possess an intrinsic stratification program of functional importance, not likely to result from promiscuous gene expression. We speculate that the maintenance of the thymic tridimensional epithelial network requires a fine balance between stratification and EMT, which could be regulated by the ZEB/miR-200 double-negative feedback loop. In this context, cultured hTECs represent an insightful system to better understand the mechanisms governing epithelial stratification and plasticity