The critical pancreatic transcription factor Pdx1 is expressed throughout the pancreas early but enriched in insulin-producing ␤ cells postnatally. Previous studies showed that the 5 conserved promoter regions areas I and II (Pdx1 PB ) direct endocrine cell expression, while an adjacent region (Pdx1 XB ) containing conserved area III directs transient ␤-cell expression. In this study, we used Cre-mediated lineage tracing to track cells that activated these regions. Pdx1 PB Cre mediated only endocrine cell recombination, while Pdx1 XB Cre directed broad and early recombination in the developing pancreas. Also, a reporter transgene containing areas I, II, and III was expressed throughout the embryonic day 10.5 (E10.5) pancreas and gradually became ␤ cell enriched, similar to endogenous Pdx1. These data suggested that sequences within area III mediate early pancreas-wide Pdx1 expression. Area III contains a binding site for PTF1, a transcription factor complex essential for pancreas development. This site contributed to area III-dependent reporter gene expression in the acinar AR42J cell line, while PTF1 specifically trans-activated area III-containing reporter expression in a nonpancreatic cell line. Importantly, Ptf1a occupied sequences spanning the endogenous PTF1 site in area III of E11.5 pancreatic buds. These data strongly suggest that PTF1 is an important early activator of Pdx1 in acinar and endocrine progenitor cells during pancreas development.The characterization of genetic regulatory elements and the transcription factors operating through these elements during pancreas development contributes to our understanding of insulin-producing ␤-cell formation. Perturbations in the transcription factors that bind important regulatory control elements can lead to defective pancreatic function and diabetes (2,19,41,48). Proper development of the endocrine and exocrine compartments of the pancreas requires several characterized transcription factors, including pancreas transcription factor 1a (Ptf1a) and the homeodomain transcription factor pancreas and duodenum homeobox 1 (Pdx1).The pancreas transcription factor 1 complex (PTF1) was first identified as an activator of exocrine-specific genes (35) and is comprised of an acinar-cell-enriched basic helix loop helix protein (bHLH), Ptf1a (p48); a ubiquitous bHLH protein (24), HEB; and the distinct mammalian Suppressor of Hairless (RBP-J) (28) or its paralogue (RBP-L) (2). Ptf1a is expressed as early as embryonic day 9.5 (E9.5) throughout the developing pancreas and is essential for pancreas formation and function in both mouse and human (23,25,28,42). Ptf1a null mice lack a ventral pancreatic bud and show an early arrest in dorsal bud outgrowth (23). Exocrine cells do not develop and there is limited endocrine development. The endocrine cells that do form are mislocalized to the spleen (25). Pdx1 is also expressed very early in pancreas development throughout both the dorsal and ventral pancreatic buds (13,18,27). After birth, Pdx1 is expressed at high levels in the i...