Adult pancreatic β-cells are primarily generated by self-duplication rather than stem-cell differentiation. Using a genetic lineage tracing method, the study shows that pre-existing β-cells, not pluripotent stem cells, are the main source of new β-cells during adult life and after pancreatectomy in mice. This suggests that terminally differentiated β-cells retain a significant proliferative capacity in vivo, challenging the idea that adult stem cells play a major role in β-cell replenishment. The study used a tamoxifen-inducible Cre/lox system to label β-cells and found that only pre-existing β-cells and their progeny were labeled, while cells derived from non-β sources, including stem cells, were not. This method revealed that no new islets are formed during adult life, and small clusters of β-cells likely represent static mini-islets or disintegrating old islets, not stem-cell-derived islets. Further analysis showed that β-cells are generated by self-duplication of pre-existing β-cells, as evidenced by the stable frequency of labeled β-cells over time. Even after partial pancreatectomy, new β-cells were found to be HPAP+, indicating they originated from existing β-cells rather than stem cells. These findings suggest that β-cell regeneration occurs through self-duplication rather than stem-cell differentiation. The study also highlights the importance of direct lineage tracing over histological snapshots to determine the origin of cells. The method developed can be used to assess stem cell contributions in other organs and to determine the cell of origin in cancer models. The results have implications for cell-based therapies for type I diabetes, as they demonstrate the significant proliferative potential of differentiated β-cells in vivo.Adult pancreatic β-cells are primarily generated by self-duplication rather than stem-cell differentiation. Using a genetic lineage tracing method, the study shows that pre-existing β-cells, not pluripotent stem cells, are the main source of new β-cells during adult life and after pancreatectomy in mice. This suggests that terminally differentiated β-cells retain a significant proliferative capacity in vivo, challenging the idea that adult stem cells play a major role in β-cell replenishment. The study used a tamoxifen-inducible Cre/lox system to label β-cells and found that only pre-existing β-cells and their progeny were labeled, while cells derived from non-β sources, including stem cells, were not. This method revealed that no new islets are formed during adult life, and small clusters of β-cells likely represent static mini-islets or disintegrating old islets, not stem-cell-derived islets. Further analysis showed that β-cells are generated by self-duplication of pre-existing β-cells, as evidenced by the stable frequency of labeled β-cells over time. Even after partial pancreatectomy, new β-cells were found to be HPAP+, indicating they originated from existing β-cells rather than stem cells. These findings suggest that β-cell regeneration occurs through self-duplication rather than stem-cell differentiation. The study also highlights the importance of direct lineage tracing over histological snapshots to determine the origin of cells. The method developed can be used to assess stem cell contributions in other organs and to determine the cell of origin in cancer models. The results have implications for cell-based therapies for type I diabetes, as they demonstrate the significant proliferative potential of differentiated β-cells in vivo.