With a genome size of ~580 kb and approximately 480 protein coding regions, Mycoplasma genitalium is one of the smallest known self-replicating organism. The reduced genomic content of M. genitalium has led researchers to suggest that the molecular assembly represented by this organism may be a close approximation to the minimal set of genes required for bacterial growth. One challenge to its study has been that it has extremely fastidious nutrient requirements. Here, we present a flux balance analysis (FBA) approach to examine the substrate uptake requirements for M. genitalium. First, we constructed and curated a genome-scale in silico metabolic model of M. genitalium, including a biomass equation. Next, this model was validated by comparing FBA simulation predictions with experimental data for growth of various mutant strains. For instance, these include classifications of genes as essential or nonessential for growth. Throughout the reconstruction, we address particular challenges such as broad specificity of enzymes, mineral uptake, and “blocked” reactions (reactions incapable of carrying any flow under any uptake environment). The validated model is employed to predict mathematically viable uptake environments for M. genitalium. Specifically, this process is used to systematically guide the construction of chemically defined medium based on the underlying metabolic pathways. Although M. genitalium is the organism examined in this study, the approaches and tools are applicable to other small/minimal-genome organisms.