In this thesis, changes in Young's modulus of mesenchymal stem cells (MSCs) were investigated during their osteogenic differentiation on bioactive peptide nanofibers that bear triple glutamic acid sequence (EEE), a non-collagenous protein sequence of some extracellular matrix (ECM) proteins (e.g. bone sialoprotein) found in bone tissue. MSCs formed spherical cell aggregates on the osteoinductive peptide nanofibers, here also called osteospheroids, of which their cells made intensive cell-cell contacts and showed osteoblast-like cell morphology. Mechanical characterization of the osteospheroids on the peptide nanofiber hydrogel was performed using atomic force microscope (AFM) where AFM probes modified with a thin film coating of octa uorocyclobutane (C4F8) were used to measure force maps of the cells at days 3, 7 and 14 of osteogenic differentiation. Hertz Cone model, same as Sneddon, was applied to approach curves of 12 force curves per cell to calculate the Young s modulus values. As a result, an increasing pattern was observed in the average Young's modulus of rMSCs on the peptide nanofibers throughout the osteogenic differentiation. Mineral deposition by the cells on the peptide nanofibers was checked by Alizarin red staining and a large amount of mineral deposition by the osteospheroids was observed, proving that an efficient osteogenic differentiation of the rMSCs happens on the osteoinductive peptide nanofibers. In the literature, a gradual decrease is shown in AFM measured average Young's modulus values of adhered MSCs throughout their osteogenic differentiation. When gelatin-coated glass was used as substrate instead of the peptide nanofibers, rMSCs showed a decreasing pattern in their Young's moduli, similarly to the literature, due to osteogenic differentiation. However, there was no significant calcium mineral deposition on the gelatin group, even until day 14 of osteogenic differentiation, indicating that a limited or a slower progression of osteogenic differentiation of rMSCs is present on the gelatin compared to rMSCs on the osteoinductive peptide nanofibers. A correlation between the observed increase in average Young's moduli of osteospheroid rMSCs and their mineral production on the peptide nanofibers may suggest a predominant role of biomineralization on the cell mechanics.