OBJECTIVE-In vitro expansion of β-cells from adult human islets could solve the tissue shortage for cell replacement therapy of diabetes. Culture of human islet cells typically results in <16 cell doublings and loss of insulin expression. Using cell lineage tracing, we demonstrated that the expanded cell population included cells derived from β-cells. Understanding the molecular mechanisms involved in β-cell fate in vitro is crucial for optimizing expansion and redifferentiation of these cells. In the developing pancreas, important cell-fate decisions are regulated by NOTCH receptors, which signal through the hairy and enhancer of split (HES)-1 transcriptional regulator. Here, we investigated the role of the NOTCH signaling pathway in β-cell dedifferentiation and proliferation in vitro. RESEARCH DESIGN AND METHODS-Isolated human islets were dissociated into single cells. β-Cells were genetically labeled using a Cre-lox system delivered by lentiviruses. Cells were analyzed for changes in expression of components of the NOTCH pathway during the initial weeks in culture. HES-1 expression was inhibited by a small hairpin RNA (shRNA), and the effects on β-cell phenotype were analyzed. RESULTS-Human β-cell dedifferentiation and entrance into the cell cycle in vitro correlated with activation of the NOTCH pathway and downregulation of the cell cycle inhibitor p57. Inhibition of HES-1 expression using shRNA resulted in significantly reduced β-cell replication and dedifferentiation. CONCLUSIONS-These findings demonstrate that the NOTCH pathway is involved in determining β-cell fate in vitro and suggest possible molecular targets for induction of β-cell redifferentiation following in vitro expansion.