This article reviews recent developments and applications of two beam systems (focused ion beam [FIB] and scanning electron microscope [SEM]) for in situ characterization and manipulation of material at the micro- and nanoscale. In these applications, the sample may be manipulated, ion milled, mechanically or electrically excited, and its temperature varied from above room temperature to cryogenic levels. FIB-SEM instruments offer new opportunities for in situ characterization by enabling localized exposure of surface layers within the high vacuum microscope chamber environment (especially in conjunction with cryogenic cooling of the bulk sample), through experiments that require either highly accurate material removal or localized material addition through beam-induced gas deposition, and by using micro- and nano-manipulation technologies for probing or positioning. This article describes the current state of the art of this experimental methodology and provides case studies in the areas of cryogenic, electrical, and mechanical characterization.