Rationale: Mesenchymal stem cells have been isolated from adult bone marrow, peripheral blood, adipose tissue, trabecular bone, articular synovium and bronchial submucosa. Objectives: We hypothesized that the lungs of premature infants undergoing mechanical ventilation contain fibroblast-like cells with features of mesenchymal stem cells. Methods: Tracheal aspirate fluid from mechanically-ventilated premature (< 30 wk gestation) infants 7 days of age was obtained from routine suctioning and plated on plastic culture dishes. Measurements and Main Results: Eleven of twenty patients studied demonstrated fibroblast-like cells, which were identified as early as 6 h after plating. Cells were found to express the mesenchymal stem cell markers STRO-1, CD73, CD90, CD105 and CD166, as well as CCR2b, CD13, prolyl 4-hydroxylase and -smooth muscle actin. Cells were negative for the hematopoietic and endothelial cell markers CD11b, CD31, CD34 or CD45. Tracheal aspirate monocyte chemoattractant protein-1 levels were 9-fold higher in aspirates in which fibroblastlike cells were found, and cells demonstrated chemotaxis in response to monocyte chemoattractant protein. Placement of cells into appropriate media resulted in adipogenic, osteogenic and myofibroblastic differentiation. Patients from whom mesenchymal stem cells were isolated tended to require more days of mechanical ventilation and supplemental oxygen. Conclusions: Together, these data demonstrate that tracheal aspirate fluid from premature, mechanically-ventilated infants contains fibroblasts with cell markers and differentiation potential typically found in mesenchymal stem cells.