The extracellular matrix (ECM) is a nanostructured environment that provides chemical, mechanical, and topographical stimuli for various cellular functions. Here, we introduce the application of laser interference lithography (LIL) to generate hexagonally arranged gold nanostructures of three different dimensions on silicon to study the effect of feature dimensions on human adipose-derived stem cells (hADSC) in terms of adhesion, growth, and differentiation. Self-assembled monolayers (SAM) were used to passivate the background silicon surface with a long-chain polyethylene glycol (PEG), whereas the gold nanostructures were activated with mercaptoundecanoic acid (MUDA) to direct protein adsorption and cell adhesive structures to them, only. It was possible to show that the size and distance of the nanostructures affected the spreading of hADSC with a decrease of cell size with the increase of feature dimensions, which corresponded also to the expression of focal adhesions and presence of the small GTPase RhoA. Effects of these early events, related to outside-in signal transduction, were visible by an enhanced cell growth on smaller feature dimensions and distinct effects on cell differentiation. Because of the precise control of chemical and topographical cues, the presented system offers great potential to study effects of material topography on stem cell behavior, which may pave the way for applications in tailoring surfaces of implants and tissue engineering scaffolds.