Tooth development is precisely controlled by epithelium-mesenchyme interactions, coordinated signaling pathways and associated transcription factors. Although the processes involved in tooth development are well established, details of the cellular and molecular mechanisms that control tooth development are not fully understood. One of the primary unknown mechanisms is the regulation of dental epithelial stem cells (DESCs), including DESC specification, proliferation and differentiation. In this dissertation, I have addressed this gap in knowledge by studying the role of Pituitary homeobox 2 (Pitx2) and Iroquois 1 (Irx1) in teeth at the cellular and molecular level in mice. PITX2 contains mutations of which are associated for Axenfeld-Rieger syndrome (ARS) in humans and is also required for early tooth development. All the background knowledge is included in Chapter I. In Chapter II, I describe the conditional ablation of Pitx2 in the dental epithelium using a Krt14Cre driver line (Pitx2cKO mice). Knocking out Pitx2 in teeth led to delayed epithelial invagination at bud stage and disruption of tooth morphogenesis at cap stage. At the cellular level, Pitx2 mediates DESC differentiation, daughter cell proliferation in bud stage tooth and regulates enamel knot formation in cap stage tooth. At the molecular level, Pitx2 acts as an upstream regulator of the sonic hedgehog (Shh) signaling pathway by regulating the expression of Shh in the dental epithelial signaling center during early tooth development. In addition, I demonstrated that Pitx2 directly controls the transcription of Irx1. In Chapter III, I determined the cellular and molecular mechanisms of Irx1 in mice. Irx1 general knockout mice were generated by replacing the entire Irx1 gene body with a LacZ reporter gene. Irx1 null mice are neonatal lethal and this lethality is due to pulmonary immaturity with defective surfactant protein secretion. In teeth, Irx1 is expressed in the outer enamel epithelium (OEE) and stratum reticulum (SR) and mediates DESC to OEE and SR differentiation through regulation of Forkhead box protein J1 (Foxj1) and Sex determining region Y-box9 (Sox9). In summary, I identified a Pitx2-Irx1 regulatory network that controls DESC differentiation in teeth, which provided the field with a better understanding of tooth development and tooth regeneration.