As a therapy for type I diabetes, islet transplantation provides clear benefits in terms of increased insulin-independence and a reduced risk of hypoglycemia. However, a critical shortage of donor pancreata means that few can benefit from this approach. The ex vivo expansion of human β-cells prior to transplantation could ameliorate this problem, however, attempts to grow large numbers of β-cells that retain their native phenotype have thus far failed. Recent lineage tracing studies suggest that this problem is due to the inherent tendency of cultured human β-cells to undergo a process reminiscent of epithelial-to-mesenchymal transition (EMT). EMT describes a highly complex process that culminates in a loss of epithelial cell polarity, severance of intercellular adhesive junctions and the acquisition of a highly motile mesenchymal phenotype. Interestingly, recent evidence suggests that a transient EMT-like process may also contribute to the delamination of endocrine progenitors and subsequent islet neogenesis. The inherent susceptibility of cultured human β-cells to EMT, and the potential involvement of this process during islet neogenesis, raises important questions as to how this process is triggered and subsequently regulated. The primary purpose of this review is to describe those factors, pathways or processes that are complicit in inducing or regulating the mesenchymal transition of human β-cells. This includes addressing the role of the extracellular matrix, the contribution of select signaling pathways, and the regulatory function of microRNAs. We propose that manipulation of these cues and pathways offers the greatest potential for restoring β-cell function after ex vivo expansion.