Previous studies have demonstrated that transcription of the lymphoid enhancer binding factor 1 (Lef1) gene is upregulated in submucosal gland progenitor cells just prior to gland bud formation in the developing ferret trachea. In the current report, several animal models were utilized to functionally investigate the role of LEF1 in initiating and supporting gland development in the airway. Studies on Lef1-deficient mice and antisense oligonucleotides in a ferret xenograft model demonstrate that LEF1 is functionally required for submucosal gland formation in the nasal and tracheal mucosa. To determine whether LEF1 expression was sufficient for the induction of airway submucosal glands, two additional model systems were utilized. In the first, recombinant adeno-associated virus was used to overexpress the human LEF1 gene in a human bronchial xenograft model of regenerative gland development in the adult airway. In a second model, the LEF1 gene was ectopically overexpressed under the direction of the proximal airway-specific CC10 promoter in transgenic mice. In both of these models, morphometric analyses revealed no increase in the number or size of airway submucosal glands, indicating that ectopic LEF1 expression alone is insufficient to induce submucosal gland development. In summary, these studies demonstrate that LEF1 expression is required, but in and of itself is insufficient, for the initiation and continued morphogenesis of submucosal glands in the airway.