Pioneer transcription factors (TFs) initiate chromatin remodeling, which is crucial for gene regulation and cell differentiation. In this study, we investigated how the sequential expression of neurogenin 3 (NGN3) and NEUROD1 affects their pioneering functions during pancreatic endocrine differentiation. Using a genetically engineered mouse model, we mapped NGN3-binding sites, confirming the pivotal role of this molecule in regulating chromatin accessibility. The pioneering function of NGN3 involves dose tolerance, and low doses are sufficient. Although NEUROD1 generally acts as a conventional TF, it can assume a pioneering role in the absence of NGN3. The sequential expression of NeuroD1 and Ngn3 predominantly drives α cell generation, which may explain the inefficient β cell induction observed in vitro. Our findings demonstrate that pioneer activity is dynamically shaped by temporal TF expression and inter-TF interactions, providing insights into transcriptional regulation and its implications for disease mechanisms and therapeutic targeting and enhancing in vitro differentiation strategies.