Chronic lung disease of prematurity (CLD) is a frequent sequela of premature birth and oxygen toxicity is a major associated risk factor. Impaired alveolarization, scarring and inflammation are hallmarks of CLD. Mast cell hyperplasia is a feature of CLD but the role of mast cells in its pathogenesis is unknown. We hypothesized that mast cell hyperplasia is a consequence of neonatal hyperoxia and contributes to CLD. Additionally, mast cell products may have diagnostic and prognostic value in preterm infants predisposed to CLD. In order to model CLD neonatal wild type and mast-cell-deficient mice were placed in an O2 chamber delivering hyperoxic gas mixture (FiO2 of 0.8) (HO) for 2 weeks and then returned to room air (RA) for an additional 3 weeks. Aged matched controls were kept in RA (FiO2 of 0.21). Lungs from HO mice had increased numbers of mast cells, alveolar simplification and enlargement and increased lung compliance. Mast-cell-deficiency proved protective by preserving airspace integrity and lung compliance. The mast cell mediators β-hexosaminidase (β-hex), histamine, and elastase increased in the bronchoalveolar lavage fluid of HO wildtype mice. Tracheal aspirate fluids (TAs) from oxygenated and mechanically ventilated preterm infants were analyzed for mast cell products. In TAs from infants with confirmed cases of CLD, β-hex was elevated over time and correlated with FiO2. Mast cell exosomes were also present in the TAs. Collectively, these data show that mast cells play a significant role in hyperoxia-induced lung injury and their products could serve as potential biomarkers in evolving CLD.