We evaluated the feasibility of using molecular markers to detect nonesmall-cell lung cancer (NSCLC) lymph node metastasis in endobronchial ultrasound-guided transbronchial needle aspiration samples. Thirty-three NSCLC patients and 17 control subjects were included. Cytokeratin-19, carcinoembryonic antigen, and epithelial cell adhesion molecule identified NSCLC lymphatic involvement. This might be important to improve the negative predictive value and accurately subtype these samples. Introduction: Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) holds promise for accurate examination of mediastinal lymph nodes in NSCLC patients. However, it is not always possible to achieve a definitive diagnosis or subtype all cases. We aimed to evaluate the role of EBUS-TBNA combined with quantitative reverse transcription polymerase chain reaction (qRT-PCR) and flow cytometry (FCM) to assess tumor-associated antigens and immune responses to identify metastases and pathological patterns in lymph node aspirates. Patients and Methods: EBUS-TBNA samples from patients with NSCLC (n = 33) and nonmalignant diseases (n 17) were prospectively collected. Cytokeratin 19 (CK-19), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EPCAM), sialyl-Lewisx, CD44, and the immune compartment were analyzed using qRT-PCR and FCM. Results: In the NSCLC patients, the epithelial cell compartment was significantly increased (30.8\% vs. 12\% CD45(-) CK-19(+) cells) and showed brighter CK19 staining than controls (P=.039) using FCM. Carcinoembryonic antigen was exclusively expressed by the NSCLC epithelial compartment (35\% of the cases) and absent in controls. The NSCL Cimmune compartment showed an increased monocyte population (P=.04), and decreased lymphocyte subpopulations, anticipating a disruption in the distribution of myeloid and lymphoid immune cells. Quantitative reverse transcription polymerase chain reaction showed that CK-19, CEA, and EPCAM transcripts were significantly higher in NSCLC. A positive correlation between the primary tumor lesion size and EPCAM (rho=0.476; P=.005), CK-19 (rho=0.594; P=.001), and CEA (r=0.394; P=.023) was also found. Conclusion: The identification of CK-19, CEA, and EPCAM in EBUS-TBNA samples using FCM and qRT-PCR is feasible and might further aid in the detection of NSCLC lymph node metastasis.