The basic helix-loop-helix transcription factor Neurogenin 3 (NGN3) controls endocrine cell fate specification in uncommitted pancreatic progenitor cells. Ngn3-deficient mice do not develop any islet cells and are diabetic. All the major islet cell types, including insulin-producing beta-cells, derive from Ngn3-positive endocrine progenitor cells. Therefore, the characterization of this population of immature cells is of particular interest for the development of novel strategies for cell replacement therapies in type 1 diabetes. To explore further the biology of islet progenitor cells we have generated a mouse in which Ngn3-expressing cells are labeled with the enhanced yellow fluorescent protein (EYFP) using a knock-add-on strategy. In this approach, the EYFP cDNA is introduced into the 3'-untranslated region of the proendocrine transcription factor, Neurogenin 3, without deleting any endogenous coding or regulatory sequences. In Ngn3(EYFP/+) and Ngn3(EYFP/EYFP) mice, the EYFP protein is targeted to Ngn3-expressing progenitors in the developing pancreas, and islets develop normally. Islet progenitors can be purified from whole embryonic pancreas by fluorescence-activated cell sorting from Ngn3(EYFP/+) mice and their development can be monitored in real time in pancreas explant cultures. These experiments showed that endocrine progenitors can form de novo and expand, in vitro, in the absence of signals from the surrounding mesenchyme, suggesting that endocrine commitment is a default pathway. The Ngn3(EYFP) mice represent a valuable tool to study islet cell development and neogenesis in normal and diabetic animals as well as for the determination of the conditions to generate beta-cells in vitro.