G A A b st ra ct s Notch in Neurog3+ cells reduced expression of Neurog3 and its bHLH target, NeuroD1, suggesting that allocation to a goblet versus EEC cell fate results from repression of bHLH proteins. The aim of this study was to determine if the cell fate of Neurog3+ cells depends on the level of Neurog3 expression. We obtained two lines of mice with different mutant Neurog3 alleles that expressed EGFP from the Neurog3 locus. One mutant, Neurog3EGFP was a null allele with EGFP replacing the Neurog3 coding sequence. For the second allele, (Neurog3addon), EGFP sequences were inserted in-frame with Neurog3 without disrupting the Neurog3 coding sequence. The transgenic lines enabled us to study the fate EGFP+ cells with two null alleles Neurog3EGFP/EGFP, one normal allele (Neurog3EGFP/+), and two normal Neurog3 expressing alleles, Neurog3addon/+. As expected, mice with two null alleles died shortly after birth, with no EECs. In Neurog3addon mice, about 60% of the EGFP+ cells expressed ChgA with the remainder in the lower crypt with a flask-like shape, suggesting they were EEC precursors that had yet to express ChgA. However, EGFP+ cells did not coexpress Muc2 or Lysozyme, markers of nonendocrine secretory lineage differentiation. In contrast, 17% of EGFP+ cells from Neurog3EGFP/+ mice stained for Muc2, with 37% stained for ChgA. The remaining EGFP+ cells appeared to be EEC precursors in the lower crypts. Neurog3EGFP/+ mice showed reduced expression of Neurog3 and its downstream target, NeuroD by qPCR, suggesting that reduced Neurog3 expression from a single Neurog3 allele may account for increased goblet cell fate. RNAseq of sorted EGFP+ cells from Neurog3EGFP/ + mice revealed that the goblet cell genes Muc2 and TFF3 were among the six most highly expressed transcripts and were enriched up to 200 fold compared to sorted, mature serotonin cells from Tph1-CFP mice. EGFP+ cells from Neurog3EGFP/+ mice show enriched expression of gut hormones and NeuroD1 (up to 150-fold) versus nonendocrine cells. Our studies further confirm that Neurog3+ cells are multipotential cells with Neurog3EGFP+ cells giving rise to a mixed population of EEC and goblet cells. High levels of Neurog3 drive EEC cell fate whereas reduced Neurog3 expression results in increased goblet cell number. We conclude that allocation between endocrine and nonendocrine secretory lineage cell fate is regulated by the level of Neurog3 expression.