The structure of the Fe3O4(001) surface was studied using surface X-ray diffraction in both ultra-high vacuum, and higher-pressure environments relevant to water-gas shift catalysis. The experimental X-ray structure factors from the 2x2 R45° reconstructed surface are found to be in excellent agreement with the recently proposed subsurface cation vacancy (SCV) model for this surface (Science 346 (2014), 1215). Further refinement of the structure results in small displacements of the iron atoms in the first three double layers compared to structural parameters deduced from LEED I-V experiments and DFT calculations. An alternative, previously proposed structure, based on a distorted bulk truncation (DBT), is conclusively ruled out. The lifting of the 2×2 R45° reconstruction upon exposure to water vapor in the mbar pressure regime was studied at different temperatures under flow conditions, and a roughening of the surface was observed. Addition of CO flow did not further change the roughness perpendicular to the surface but decreased the lateral correlations.