Purpose: Mediastinal lymphadenopathy (ML) is a cause for concern, especially in patients with previous malignancy. We report our experience with the use of endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) with immunocytochemical stains in patients being evaluated for ML. Methods: Retrospective analysis of patients with ML of unknown origin who underwent EUS-FNA. On-site evaluation was performed by experienced cytologist, and special immunocytochemical stains were requested as indicated. Results: A total of 116 patients were included, and a total of 136 mediastinal LN were sampled. Prior malignancy was present in 45%. The most common site of examined lymph node (LN) were subcarinal (76%, 103 LN). The median long and short axis diameters were 28 mm and 13 mm, respectively. FNA was read on-site as malignant, 21 (16%); benign, 100 (76.9%); suspicious, six (4%); atypical, 3 (2%); and inadequate sample, six (4%). Sixty-four LN were deferred for additional studies; 22 for immunocytochemical and 26 for Gimesa (GMS) stain and 21 for flow cytometry. Final FNA read was malignant in 28 (21%), benign in 103 (76%), suspicious in three (2%), and atypical in two (1%). Metastatic malignancies disclosed included Hodgkin′s and Non-Hodgkin′s lymphoma, melanoma, hepatoma, breast, lung, colon, renal, endometrial, Fallopian tube, and unknown carcinoma. The sensitivity, specificity, and accuracy of the final FNA read to predict malignancy were 100%. Conclusion: EUS-guided FNA with additional ancillary studies is useful in disclosing metastatic ML from a variety of neoplasms. Due to its safety and accuracy profile, it should be considered the test of choice in evaluating abnormal ML in appropriately selected patients.