Thymic epithelial cells (TECs) are crucial to the ability of the thymus to generate T cells for the adaptive immune system in vertebrates. However, no in vitro system for studying TEC function exists. Overexpressing the transcription factor FOXN1 initiates transdifferentiation of fibroblasts into TEC-like cells (iTECs) that support T-cell differentiation in culture or after transplant. In this study, we have characterized iTEC programming at the cellular and molecular level in mouse to determine how it proceeds, and have identified mechanisms that can be targeted for improving this process. These data show that iTEC programming consists of discrete gene expression changes that differ early and late in the process, and that iTECs upregulate markers of both cortical and medullary TEC (cTEC and mTEC) lineages. We demonstrate that promoting proliferation enhances iTEC generation, and that Notch inhibition allows the induction of mTEC differentiation. Finally, we show that MHCII expression is the major difference between iTECs and fetal TECs. MHCII expression was improved by co-culturing iTECs with fetal double-positive T-cells. This study supports future efforts to improve iTEC generation for both research and translational uses.

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