Hydrogen fuel cells in fuel cell electric vehicles (FCEVs) are a promising technology to reduce, and eventually eliminate, carbon dioxide emissions from transportation. The Pt nanoparticles used to catalyze the fuel cell’s electrochemical reactions are an important limiting factor because at present levels, the cost of the Pt catalyst will prevent widespread adoption of FCEVs. Catalysts must be developed to reduce the amount of Pt while meeting vehicle power demands even after many years of use. Strategically improving catalysts requires detailed and statistically robust characterization of their microscopic structure to understand the connections between catalyst synthesis, structure, performance, and durability. This dissertation presents the development and application of (scanning) transmission electron microscopy ((S)TEM) techniques to guide advancement of catalysts through nanostructural characterization. We develop a robust strain mapping technique for complex catalyst specimens. We deploy a new exit wave power cepstrum (EWPC) transform to nanobeam electron diffraction (NBED) patterns to enable precise, high-throughput, dose-efficient strain measurement. This approach is suitable for statistically representative measurements of many particles without special requirements such as zone-axis orientation. We apply this strain mapping technique to core-shell Pt-Co nanoparticles in combination with a continuum elastic theory model and demonstrate two mechanisms contributing to the relaxation of strain at the catalyst surface: lattice dislocations and Poisson expansion due to the spherical geometry. Comparison with electrochemical measurements suggests that the geometrical Poisson relaxation accounts for the activity of catalysts with thin shells, but catalysts with thick shells experience additional activity loss from dislocation-driven relaxation. We then turn to the larger-scale catalyst structure, investigating the impact of porous carbon support morphology, local reactant transport, and catalyst durability. Using statistical analysis of STEM images, we compare Pt and Pt-Co catalysts on porous and solid carbon supports. Comparison of 3D tomographic images and electrochemical accessibility measurements indicated that carbon pores prevent ionomer adsorption for particles embedded within them, improving the catalyst activity, while allowing proton access through condensed water. By comparing images and composition maps before and after electrochemical stability tests, we find that porous carbon supports suppress Pt particle coalescence, accounting for improved overall durability.