In this study, cell-material adhesive strength and cellular mechanical properties were measured using atomic force microscopy (AFM) to track cell attachment and osteogenic differentiation. First, chitosan substrates were treated with simulated body fluid (SBF) for various periods, resulting in substrates with different osteoconductivity. The X-ray diffraction (XRD), energy-dispersive X-ray spectroscopy (EDS) and in vitro tests revealed that the biomimeticity and osteoconductivity of substrates increased with increasing time of SBF treatment. When the SBF immersion exceeded 14 days, the chitosan substrates exhibited their highest biocompatibility and osteoconductivity. AFM measurements indicated specifically high adhesive forces between SBF-treated chitosan and osteogenic cells, causing better cell attachment. The results demonstrate that cell adhesion was controlled by cell-material adhesive strength, which were in turn controlled via the SBF treatment time. The adhesive strength between cells and material also accounted for the chitosan substrates' specific selectivity toward osteogenic cells. A two-step increase in mechanical strength was observed for the nucleus and cytoplasm of osteogenic cells. The results indicate that through the use of AFM, the real-time cell-material interforce and cellular mechanics can be identified. The adhesive strength was positively correlated to the cell attachment, and the second increase in the Young's modulus of nucleus and cytoplasm was correlated to early osteogenic differentiation.