Cells respond profoundly to the mechanical rigidity and three-dimensional nanotopology of substrates, as well as the spatial and temporal arrangements of extracellular cues. We summarize the latest developments in probing and engineering biocompatible nanofibrous extracellular environments at the cell and molecular level for applications in tissue engineering and biological research. This will, in turn, guide further development of three-dimensional nanofibrous scaffolds in order to elicit specific cellular responses for relevant applications.