The development of air-filled respiratory organs is crucial for survival. We have combined live imaging with genetics to dissect airway maturation. Initially, a secretion burst deposits proteins into the nascent tracheal tubes. Solid material is then rapidly cleared and shortly thereafter liquid is removed from the lumen. The mechanisms underlying the precise spatial and temporal regulation of epithelial activities during airway maturation are unknown. We have used a tracheal specific driver and ~20000 transgenic UAS-RNAi strains to first describe all protein-coding genes involved in the process. To identify the developmental regulators of airway maturation, we preselected about 600 genes encoding putative regulators and further classified these genes into 12 groups based on the defects in tube morphologies, apical secretion and protein clearance events caused by their tracheal inactivation. The combination of this phenotypic analysis with the data from protein interaction databases reveals new gene regulatory networks controlling airway maturation.