Much of our knowledge of islet physiology stems from rodent studies. However, we now know that marked discrepancies exist between rodent and human islets in regard to (for example) architecture, hormone secretion, and islet cell transcription factor (TF) expression, demonstrating potential limitations in assuming that rodent models entirely mimic human islet physiology and disease. An example of such a discrepancy lies in the islet β cell enriched MAFA TF, a fundamentally important protein to these cells in postnatal rodents. Thus, the MAFA protein is barely detectable in human islet β cells until ~9 years of age, whereas this TF is first detected developmentally and then produced throughout the lifespan of rodent insulin+ cells. We propose that humans have at least two postnatal, age-dependent MAFA-producing β cell populations capable of maintaining euglycemia: juvenile β cells (i.e. <~9 years old) with little MAFA (i.e. MAFALow) and post-juvenile β cells with robust MAFA (MAFAHigh). In fact, independent studies have established distinct molecular and functional properties of these human cell populations. Here we will examine the impact of MAFA on human β cells and hypothesize that this TF plays an essential role in regulating insulin secretion in adults. Our analysis in the first aim will be conducted using the β-like cells produced from both human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) (collectively termed human pluripotent stem cells, or hPSCs). Here we will determine how knockout and over-expression of MAFA influences β cell maturation and function, appreciating that their greatly improved glucose-stimulated insulin secretion properties parallel enhanced MAFA expression after transplantation into immunocompromised NSG mice. In addition, we will investigate how a pathogenic variant of MAFA (Serine (S) 64 -> Phenylalanine (F)) that prominently increases its protein stability affects human β cell activity. Notably, MAFAS64F predisposes subjects to either adult-onset diabetes or insulinomatosis (i.e. non-syndromic insulin-producing β cell tumors) in a gender-biased manner. We generated a mouse model harboring this mutation in the endogenous MafA gene, and our results show glucose intolerance in males and improved glucose clearance in females, mimicking the findings in human subjects. Significantly, dysfunction in male mice was associated with premature cellular aging and senescence. Recently, independent reports have linked pathologic, senescent β cell populations to type 1 diabetes and type 2 diabetes islet dysfunction. Our experimentation in the second aim will define the molecular and functional consequences of MAFAS64F in human β cells. Our overall focus on human islet cells is viewed as innovative, as well as the combined use of human stem cell derived β-like cells and human islets for obtaining novel, physiologically relevant mechanistic insights.