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This article reports a novel method to effectively modify the surface stiffness for the differentiation of stem cell growth. To achieve large range of surface hardness, focused electron beam is first employed to radiate hydrogen silsesquioxane (HSQ) film. With different degrees of curing caused by certain e-beam exposure, the HSQ demonstrates various Young’s modulus from 0.5 to 2 GPa, measured by an atomic force microscope. Fourier transform infrared spectra were used to investigate the origin of the stiffness change, which is due to the e-beam irradiation induced network formation inside HSQ. The novel technique possesses a number of advantages such as precision control of stiffness in a broad matrix with high spatial resolution. It also offers a good opportunity to define the geometry shape with a constant stiffness in nanometer scale.