Understanding how biomineralization occurs in the extracellular matrix (ECM) of bone cells is crucial to the development of a successfully engineered bone tissue scaffold, and to date there has not been a well-established method for the quantitative examination of bone mineralization in situ. We investigated the mechanical properties of MC3T3-E1 osteoblast-like cells and the crystalline properties of their biomineralized ECM in vitro using shear modulation force microscopy (SMFM), confocal laser scanning microscopy (CLSM), synchrotron X-ray diffraction (XRD), and Fourier-transform infrared spectroscopy (FTIR). The elastic modulus of the mineralizing cells increased at time points corresponding to mineral production, whereas that of the non-mineralizing cells did not vary significantly over time. CLSM showed a restructuring of the F-actin fiber network of mineralizing cells with time, which indicates remodeling activities in the cytoskeleton and was not seen in the non-mineralizing cells. Both XRD and FTIR showed that the mineralizing subclone produced hydroxyapatite in situ and that the non-mineralizing subclone was in fact weakly biomineralizing.