The olfactory epithelium (OE) regenerates after injury via two types of tissue stem cells: active globose cells (GBCs) and dormant horizontal basal cells (HBCs). HBCs are roused to activated status by OE injury when P63 levels fall. However, an in-depth understanding of activation requires a system for culturing them that maintains both their self-renewal and multipotency while preventing spontaneous differentiation. Here, we demonstrate that mouse, rat, and human HBCs can be cultured and passaged as P63+ multipotent cells. HBCs in vitro closely resemble HBCs in vivo based on immunocytochemical and transcriptomic comparisons. Genetic lineage analysis demonstrates that HBCs in culture arise from both tissue-derived HBCs and multipotent GBCs. Treatment with retinoic acid induces neuronal and non-neuronal differentiation and primes cultured HBCs for transplantation into the lesioned OE. Engrafted HBCs generate all OE cell types, including olfactory sensory neurons, confirming that HBC multipotency and neurocompetency are maintained in culture.