The thymus plays a key role in central tolerance through maturation and selection of thymocytes into functional and non-self-reactive T cells. Within the thymus, the primary functional cells are the thymic epithelial cells (TECs), which are further divided between the cortex, responsible for positive selection of functional thymocytes, and the medulla, responsible for negative selection of self-reactive T cells. While there is increasing knowledge of the heterogeneity within TECs, and in particular TECs within the medulla (mTECs), the precise mechanisms underlying the regulatory control leading to this heterogeneity remains largely unknown. Recent studies have identified members of the Grainyhead-like (Grhl) transcription factor family, specifically Grhl1 and Grhl3, to be associated with multiple facets of thymus development. Here, we focus on Grhl3, identifying it as more specific to early mTEC development than other members of the family and utilize a conditional knockout to reveal two opposing effects within the adult thymus. Furthermore, we identify perturbed development in the embryonic thymus, providing some evidence of a possible origin for the perturbations in the adult. In summary, this dissertation provides insight into the role of the Grainyhead-like family within the thymus and the regulatory control that it exerts.