The mammalian airways are lined by a continuous epithelial layer that is maintained by diverse populations of resident multipotent stem cells. These stem cells are responsible for replenishing the epithelium both at homeostasis and following injury, making them promising targets for stem cell and genetic-based therapies for a variety of respiratory diseases. However, the mechanisms that regulate when and how these stem cells proliferate, migrate, and differentiate remains incompletely understood. Here, we find that the high mobility group (HMG) domain transcription factor Lef-1 regulates proliferation and differentiation of mouse tracheal basal cells. We demonstrate that conditional deletion of Lef-1 stalls basal cell proliferation at the G1/S transition of the cell cycle, and that Lef-1 knockout cells are unable to maintain luminal tracheal cell types in long-term air-liquid interface culture. RNA sequencing analysis revealed that Lef-1 knockout (Lef-1KO) results in downregulation of key DNA damage response and cell cycle progression genes, including the kinase Chek1. Furthermore, chemical inhibition of Chek1 is sufficient to stall basal cell self-renewal in a similar fashion as Lef-1 deletion. Notably, the cell cycle block imposed by Lef-1KO in vitro is transient and basal cells eventually compensate to proliferate normally in a Chek1-independent manner. Finally, Lef-1KO cells were unable to fully regenerate tracheal epithelium following injury in vivo. These findings reveal that Lef-1 is essential for proper basal cell function. Thus, modulating Lef-1 function in airway basal cells may have applications in regenerative medicine. © AlphaMed Press 2021 SIGNIFICANCE STATEMENT: Airway basal cells (BCs) are the primary progenitor cells of the conducting airway epithelium and thus are promising targets for durable genetic- and cell-based therapies of lung diseases such as cystic fibrosis. However, the mechanisms that govern their regenerative abilities are not fully understood. The authors’ show that Lef-1, an effector of Wnt signaling, is critical for BC cell cycle progression through G1/S and influences BC differentiation in vitro. Loss of Lef-1 significantly reduces the capacity of BC to regenerate the tracheal epithelium in vivo following injury. Manipulating Lef-1 expression in BCs may provide opportunities to modulate the regenerative capacity of BCs and their progeny.© 2021 AlphaMed Press.