Methods of manufacturing three-dimensional semiconductor devices that may include forming a first spacer on a sidewall inside a first opening formed in a first stack structure, forming a sacrificial filling pattern on the spacer to fill the first opening, forming a second stack structure including a second opening exposing the sacrificial filling pattern on the first stack structure, forming a second spacer on a sidewall inside the second opening, removing the sacrificial filling pattern and removing the first spacer and the second spacer.