In order to gain novel insights into thymus biology, we analysed the whole transcriptome of cortical and medullary thymic epithelial cells (cTECs and mTECs) and of skin epithelial cells (ECs). Consistent with their ability to express ectopic genes, mTECs expressed more genes than other cell populations. Out of a total of 15,069 genes expressed in TECs, 25% were differentially expressed by at least 5-fold in cTECs vs. mTECs. Genes expressed at higher levels in cTECs than mTECs regulate numerous cell functions including cell differentiation, cell movement and microtubule dynamics. Many positive regulators of the cell cycle were overexpressed in skin ECs relative to TECs. Our RNA-seq data provide novel systems-level insights into the transcriptional landscape of TECs, highlight substantial divergences in the transcriptome of TEC subsets and suggest that cell cycle progression is differentially regulated in TECs and skin ECs.