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The larval lepidopteran midgut is a complex tissue system that shows significant structure-function relationships related to its roles in digestive and absorptive processes. δ-endotoxins (Cry toxins) produced by the bacterium Bacillus thuringiensis disrupt the midgut epithelium of target insects has been used extensively to control pests. However, insects, including several lepidopteran species, evolve resistance to Cry toxins which causes a great threat to their continued utility. Understanding the physiology of the midgut, including that of the stem cells which are responsible for midgut growth, development, and regeneration, may improve the sustainability of midgut-targeted control like Cry toxins. Historically, lepidopteran midgut stem cells have been distinguished from mature cells by morphology, but this is unreliable due to significant morphological variation in both mature and stem populations, including during the differentiation processes of the latter. Thus, we examined three vital markers to distinguish larval lepidopteran midgut stem and mature cell types, as well as the differentiation state of stem cells using esterase activity (Calcein AM), mitochondrial density (Mitotracker), and mitochondrial membrane potential (TMRM). We also identified the existence and the expression level of one stemness maintainer gene Escargot among different development stages of lepidopteran larvae. Our results support the use of mitochondrial properties in lepidopteran midgut cell differentiation and indicate esterase activity is an insufficient marker even combined with morphology. Further, escargot transcript patterns support further examination of the role of the protein in gut physiology, including stem maintenance. Our results provide tools for characterization and modification of physiological responses of lepidopteran midgut cells to stimuli and stresses. These in turn will aid in understanding conservation and divergence of developmental processes and development and use of pest control resources.