The origin of the thymic epithelium, i.e. the cortical (cTEC) and medullary (mTEC) epithelial cells, from bipotent stem cells through TEC progenitors and lineage-specific progeny still remains poorly understood. We sought to obtain an unbiased view of the incipient emergence of TEC subsets by following embryonic TEC development based on co-expression of EpCAM, CD80 and MHC class II (MHCII) on non-hematopoietic (CD45(-) ) thymic stromal cells in wild-type BL6 mice. Using a combination of ex vivo analysis, Re-aggregate Thymic Organ Culture (RTOC) reconstitution assays and mathematical modelling, we traced emergent lineage commitment in murine embryonic TECs. Both experimental and mathematical datasets supported the following developmental sequence: MHCII(-) CD80(-) → MHCII(lo) CD80(-) → MHCII(hi) CD80(-) → MHCII(hi) CD80(hi) TECs, whereby MHCII(hi) CD80(-) and MHCII(hi) CD80(hi) TECs bear features of cTECs and mTECs respectively. These emergent MHCII(hi) CD80(-) cTECs directly generate mature MHCII(hi) CD80(hi) mTECs in vivo and in vitro, thus supporting the asynchronous model of TEC lineage commitment. This article is protected by copyright. All rights reserved.