Mechanical behavior of metallic materials on nanoscale is characterized by using Nanoindentation and Transmission Electron Microscope (TEM) to understand the fundamental plasticity mechanisms associated with microstructural factors including dislocations. The advanced characterization techniques enable us to grasp the behavior on the nanoscale in detail. New knowledges are obtained for the plasticity initiation under the extremely high stress close to the theoretical strength in regions with defect-free matrix and pre-existing defects such as grain boundaries, in-solution elements, and dislocations. The grain boundaries act as an effective dislocation source, the in-solution elements retard a nucleation of dislocation, and the pre-existing dislocations assist a plasticity initiation. The deformation behavior associated with microstructures is also described. The dislocation structure with a certain density was observed right after indentation-induced strain burst, which is so-called “pop-in,” suggesting a dislocation avalanche upon the pop-in. It has been directly observed that the lower mobility screw dislocation causes the higher flow stress in a bcc metal. A remarkable strain softening can be understood by an increase in dislocation density based on conventional physical models. Phase stability for indentation-induced transformation depends on a constraint effect by inter-phase boundary and grain boundary.