Considerable evidence points to the critical role of the gut microbiota in physiology and disease. The administration of live microbes as a therapeutic modality is increasingly being considered. However, key questions such as how to identify candidate microorganisms and which preclinical models are relevant to recapitulate human microbiota remain largely unanswered. The establishment of a humanized gnotobiotic mouse model through the fecal microbiota transplantation of human feces into germ-free mice provides an innovative and powerful tool to mimic the human microbial system. However, numerous considerations are required in designing such a model, as various elements, ranging from the factors pertaining to human donors to the mouse genetic background, affect how microbes colonize the gut. Thus, it is critical to match the murine context to that of human donors to provide a continuous and faithful progression of human flora in mice. This is of even greater importance when the need for accuracy and reproducibility across global research groups are taken into account. Here, we review the key factors that affect the formulation of a humanized mouse model representative of the human gut flora and propose several approaches as to how researchers can effectively design such models for clinical relevance.