The related basic helix-loop-helix transcription factors neurogenin3 (Neurog3) and neurogenic differentiation 1 (NeuroD1) regulate pancreatic islet cell formation. The transient expression of Neurog3 initiates endocrine differentiation and activates its target, NeuroD1, which continues the endocrine differentiation process. Despite their distinct developmental roles, the expression of either factor can drive islet differentiation in progenitor cells. To determine whether Neurog3 and NeuroD1 function by targeting a common set of genes, we compared gene expression patterns in cells ectopically expressing these two factors using cDNA microarrays. The array data demonstrated that both factors regulated largely overlapping sets of genes, providing the molecular basis for their functional equivalence in gain-of-functions approaches. Distinct differences in the timing and level of expression of a subset of target genes, however, show that the functions of these two factors are not completely redundant. Interestingly, in addition to NeuroD1, Neurog3 also induced both NeuroD2 and NeuroD4 gene expression. NeuroD2 mRNA peaked in the embryonic pancreas during endocrine differentiation and induced endocrine differentiation in vitro. These data suggest possible redundant roles for the NeuroD1 paralogs NeuroD2 and NeuroD4 in pancreatic endocrine differentiation and their potential utility in cell-based therapies for diabetes mellitus.