Mechanical cues such as the 2-Dimensional (2D) and 3-Dimensional (3D) shape of cellular microenvironments affect several cellular processes including adhesion and proliferation. Recent studies provide controlled conditions to recapitulate the 2D and 3D microenvironments to understand the mechanisms of cellular response to these mechanical cues. Microengineering surfaces such as micropatterning techniques are widely used to modulate 2D cell shapes. Additionally, nanostructured surfaces are used to modulate 3D substrate nanotophography. By confining cells to micropatterns, cells reorganize their cytoskeleton architecture to the microenvironment by remodeling the actin and microtubule fibers. In this study, we present a micropatterning technique on nanostructured surfaces based on a maskless laser-assisted technique to study the cellular response to 3D nano-topographies in a controlled 2D microenvironment. We used a two-step dry and wet etching technique to fabricate transparent (SiO2) 3D nanostructured surfaces. Next, we micropatterned extracellular matrix proteins directly on the fabricated nanostructures by maskless micropatterning (PRIMO, Alvéole) system mounted on an inverted microscope. Briefly, UVO treatment was performed to clean the surface followed by surface incubation with Poly-L-Lysine. Subsequently, the surface was passivated with m-PEG-SVA, and a photosensitizer reagent (PLPP) applied to the surface to remove the PEG layer upon UV exposure. After UV illumination and washing the surface, nanopillars were incubated with ECM proteins (Fibronectin and Gelatin). Next, we demonstrated the 2D control of cell and nuclear shape on 3D nanostructured surfaces for an epithelial-like cell line (U2OS, ATCC) by seeding them on Fibronectin/Gelatin micropatterns of various geometries. We then used fluorescent microscopy to characterize the organization of intracellular components such as the nucleus and cytoskeletal elements. Moreover, we analyzed the spreading of cells over time, the effect of 2D micropattern geometry on cell morphology, and cytoskeleton reorganization on various 2D and 3D configurations.