Abstract: Methods of fabricating a three-dimensional semiconductor device are provided. Methods may include forming a stack structure including first layers and second layers alternately stacked on a substrate, patterning the stack structure to form at least one isolation trench, forming channel structures penetrating the stack structure and being spaced apart from the isolation trench, and forming upper interconnection lines on the stack structure to connect the channel structures to each other. An isolation trench may be formed prior to formation of the channel structures.