Cells establish and modulate their morphology and mechanics through the use of structural networks whose components range in size from a few nanometers to tens of micrometers. Over the past two decades, an exciting suite of sophisticated micro- and nanoscale technologies has emerged that permits investigators to directly probe structural and functional contributions of these components in living cells. Here we review underlying principles and recent applications of four such approaches: atomic force microscopy, subcellular laser ablation, micropatterning, and microfluidics. Together, these new tools are offering valuable insight into the molecular basis of cell structure and mechanics and revealing the remarkably broad influence of the mechanical microenvironment on many aspects of cell biology.