Episodes of graft dysfunction are frequently observed after liver transplantation and can be due to different causes requiring specific therapy. In this study the usefulness and reliability of liver transplant aspiration cytology (TAC) for differential diagnosis of liver graft dysfunction is assessed. Out of more than 1500 TACs performed, 292 TACs, taken during episodes of liver dysfunction due to retrospectively defined causes, were analyzed. Immune activation and parenchymal damage in the aspirates were determined cytologically. In 63 episodes of acute rejection, marked immune activation was present in aspirate but not in blood, with varying degrees of hepatocyte damage and cholestasis. No or only minimal immune activation was observed in 86 cases of toxic, ischemic, or septic liver damage, but considerable parenchymal damage and cholestasis were observed. In 3 cases of hepatitis slight-to-moderate immune activation with large granular lymphocytes was found in the aspirate, while 17 cases of viral infection presented with slight-to-moderate immune activation in aspirate and blood. After successful treatment the cytologic patterns normalized, except when the cause of liver dysfunction persisted. Moreover, typical patterns of parenchymal changes were found for preservation damage of the liver (n = 108), fatty degeneration (n = 3), obstructive cholestasis (n = 5), and acute arterial ischemia (n = 2). One case of moderate subcapsular hematoma was the only complication observed (less than 0.1%). Thus, liver TAC is an easy, safe, and clinically useful method for differential diagnosis of liver graft dysfunction. In particular, differentiation between acute rejection and nonimmunologic causes of dysfunction is very reliable, but hepatitis and viral infections also present distinctive patterns in liver TAC.