An early step in pancreas development is marked by the expression of the Pdx1 transcription factor within the pancreatic endoderm where it is required for the specification of all endocrine cell types. Subsequently, Pdx1 expression becomes restricted to the β-cell lineage where it plays a central role in β-cell function. This pivotal role of Pdx1 at various stages of pancreas development makes it an attractive target to enhance pancreatic β-cell differentiation and increase β-cell function. In this study, we used a newly generated zebrafish reporter to screen over 8,000 small molecules for modulators of pdx1 expression. We found four hit compounds and validated their efficacy at different stages of pancreas development. Valproic acid (VPA), a HDAC inhibitor, induces pancreatic endoderm formation, while inhibition of TGF-β signaling leads to the transdifferentiation of α-cells to β-cells. HC Toxin, another HDAC inhibitor, enhances β-cell function in primary mouse and human islets. Thus, using a whole-organism screening strategy, this study identified new pdx1 expression modulators which can be used to influence different steps in pancreas and β-cell development.