The identification of factors that regulate airway epithelial cell proliferation and differentiation are essential for understanding the pathophysiology of airway diseases. Rho-associated protein kinases (ROCKs) are downstream effector proteins of RhoA GTPase that direct the functions of cell cytoskeletal proteins. ROCK inhibition with Y27632 has been shown to enhance the survival and cloning of human embryonic stem cells and pluripotent cells in other tissues. We hypothesized that Y27632 treatment exerts a similar effect on airway epithelial basal cells, which function as airway epithelial progenitor cells. Treatment with Y27632 enhanced basal-cell proliferation in cultured human tracheobronchial and mouse tracheal epithelial cells. ROCK inhibition accelerated the maturation of basal cells, characterized by a diminution of the cell size associated with cell compaction and the expression of E-cadherin at cell–cell junctions. Transient treatment of cultured basal cells with Y27632 did not affect subsequent ciliated or mucous cell differentiation under air–liquid interface conditions, and allowed for the initial use of lower numbers of human or mouse primary airway epithelial cells than otherwise possible. Moreover, the use of Y27632 during lentivirus-mediated transduction significantly improved posttransduction efficiency and the selection of a transduced cell population, as determined by reporter gene expression. These findings suggest an important role for ROCKs in the regulation of proliferation and maturation of epithelial basal cells, and demonstrate that the inhibition of ROCK pathways using Y27632 provides an adjunctive tool for the in vitro genetic manipulation of airway epithelial cells by lentivirus vectors.