Production of high quality, disease-free seed potatoes is a lengthy, multi-year process that begins with small plantlets grown under sterile conditions of the tissue culture labs. The physiological condition and quality of these plants directly affect the subsequent stages of seed potato production, including their ability to survive transfer from in vitro conditions to greenhouse or field environments. Successful micropropagation relies heavily on the composition of the culture medium. Traditionally, commercial labs use Murashige and Skoog (MS) medium for clonal propagation of potatoes; however, the nutrients it supplies may not be sufficient for potato plants when they are grown at high density and under suboptimal lighting conditions. The aim of this study was to evaluate nutrient-related growth responses of potato plants in vitro, and to develop an improved culture medium composition for potato germplasm. We used a factorial approach to test combinations of five nutrient factors: 1) NH4NO3, 2) KNO3, 3) mesos (CaCl2, KH2PO4 and MgSO4), 4) micronutrients (B, Cu, Co, I, Mn, Mo, Zn, Fe), and 5) sucrose. We supplemented MS medium with a range of concentrations of each factor. Twenty-five medium compositions were tested using three potato cultivars—Russet Burbank, Ranger Russet, and Shepody—at a density of 20 single-node explants per Magenta vessel. The plants were observed daily, and the effects of these five factors on plant growth, quality, shoot length, leaf size, color, root development, biomass and chlorophyll content were determined. Increased mesos and, to a lesser extent, micronutrients were found to be the most important factors associated with overall improved plant quality in Russet Burbank and Ranger Russet. Increased nitrogen content resulted in greater plant growth only in Shepody. These findings will be used to develop new media formulations to ensure the best quality of micropropagated potatoes.