Organoid models are a powerful 3D stem cell technology to explore tissue (patho-)biology and development. Tissue-derived (i.e., from tissue biopsies) organoids are long-term and stably expandable while more closely recapitulating key phenotypical and functional characteristics of the tissue-of-origin than traditional 2D culture systems. Additionally, organoids can differentiate into tissue-specific cell types, for instance, following exposure to defined differentiation cues. Although prevailing in vitro cell models have deepened our understanding of mouse tooth development and biology, in vitro representations of the dental epithelium lack (the combination of) these benefits of tissue-derived organoids and are at most derived from one tooth type. Here, we describe the protocol to establish, propagate, and differentiate mouse tooth organoids from both early postnatal molar and incisor teeth. The established organoids display a dental epithelial stemness phenotype and acquire a maturation-stage ameloblast-like phenotype following differentiation.