Magnetite (Fe3O4) nanoaggregates with a flower-like morphology are considered promising materials in the field of magnetically induced hyperthermia in cancer therapy due to their good heating efficiency at low applied alternating magnetic fields. Although the structure and the magnetic state of such flower-like aggregates have been investigated previously, the mechanism that leads to the hierarchical morphology is still poorly understood. Here, we study the formation mechanism of Fe3O4 aggregates synthesized through the partial oxidation of ferrous hydroxide in the presence of poly(acrylic acid) by using cryogenic electron microscopy. The aggregates are formed through a multistep process involving first the conversion of ferrous hydroxide precursors in ∼5 nm primary particles that aggregate into ∼10 nm primary Fe3O4 crystals that finally arrange into the secondary mesocrystal structure. High-resolution electron tomography is used to show that the Fe3O4 mesocrystals are composed of ∼10 nm subunits, often showing a uniform crystallographic orientation resulting in single-crystal-like diffraction patterns. Furthermore, electron holography reveals that mesocrystals have a single magnetic domain despite polymeric interfaces between subunits being present throughout the mesocrystal. Our findings could be used to design materials with specific properties by modulating the morphology and/or magnetic state that is suitable for biomedical application.