BACKGROUND. The role of endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) in the evaluation of spindle cell and mesenchymal lesions is unclear. This study reviews the use of EUS-FNA in diagnosing intrathoracic and intra-abdominal spindle and mesenchymal cell lesions at an academic institution. METHODS. All EUS-FNA specimens with a significant spindle or mesenchymal cell component were retrieved. Follow-up was comprised of clinical correlation, chart review, or evaluation of subsequent tissue specimens, including FNAs, biopsies, and/or surgical resections. Lesions were categorized as either inflammatory/reactive or neoplastic. RESULTS. Forty-four EUS-FNA specimens were retrieved from 39 patients (21 men and 18 women with a median age of 61 years [range, 20-88 years]). Anatomic sites included 19 lymph node specimens, 15 gastrointestinal tract specimens, 7 pancreatic specimens, and 4 other anatomic site specimens. Twenty-two cases were inflammatory/reactive lesions, including 17 granulomatous lesions and 5 cases of chronic pancreatitis. Twentytwo cases were neoplastic, including 14 gastrointestinal stromal tumors, 2 smooth muscle tumors, 2 sarcomatoid carcinomas, 2 melanomas, 1 sarcoma, and 1 solitary fibrous tumor. A specific cytologic diagnosis was rendered in 30 cases (81%). Immunocytochemistry was performed on 21 neoplastic cases and contributed to the differential diagnosis in 18 cases. No false-positive findings were encountered. Three false-negative results were identified and were attributed to sampling error. CONCLUSIONS. Spindle cell neoplasms are rarely encountered on EUS-FNA. The differential diagnosis encompasses a wide variety of benign and neoplastic entities. Correlation of cytomorphology and ancillary studies yields a high diagnostic accuracy of spindle cell and mesenchymal lesions on EUS-FNA. Cancer (Cancer Cytopathol) 2011;119:37-48. © 2010 American Cancer Society.