The formation processes of iron silicide nanoparticles dependent on thermal annealing were examined by ex situ and in situ transmission electron microscopy (TEM). An Fe thin film with a thickness of ∼2 nm was deposited on a Si(100) substrate at room temperature using an electron-beam evaporation method, followed by thermal annealing at temperatures ranging from 573 to 1173 K. Ex situ TEM observations showed that pyramidal β-FeSi2 particles (1173 K) were formed via the ε-FeSi layer (573 K) and the γ-FeSi2 particle (873 K). Detailed observations by in situ TEM indicated that an amorphous Fe-Si layer was formed on the Si substrate in the as-deposited specimen. This amorphous layer was crystallized into ε-FeSi after thermal annealing and then hemispherical ε-FeSi particles together with metastable γ-FeSi2 were formed just beneath the surface of the Si substrate. With increasing annealing temperature, the ε phase changed to hemispherical γ-FeSi2 nanoparticles and finally the γ-FeSi2 particles transformed into pyramidal β-FeSi2 particles. We discuss the formation mechanism of the iron silicide nanoparticles at the atomic scale.