To determine whether glucocorticoids are involved in pancreas development, glucocorticoid treatment of rat pancreatic buds in vitro was combined with the analysis of transgenic mice lacking the glucocorticoid receptor (GR) in specific pancreatic cells. In vitro treatment of embryonic pancreata with dexamethasone, a glucocorticoid agonist, induced a decrease of insulin-expressing cell numbers and a doubling of acinar cell area, indicating that glucocorticoids favored acinar differentiation; in line with this, expression of Pdx-1, Pax-6, and Nkx6.1 was downregulated, whereas the mRNA levels of Ptf1-p48 and Hes-1 were increased. The selective inactivation of the GR gene in insulin-expressing β-cells in mice (using a RIP-Cre transgene) had no measurable consequences on β- or α-cell mass, whereas the absence of GR in the expression domain of Pdx-1 (Pdx-Cre transgene) led to a twofold increased β-cell mass, with increased islet numbers and size but normal α-cell mass in adults. These results demonstrate that glucocorticoids play an important role in pancreatic β-cell lineage, acting before hormone gene expression onset and possibly also modulating the balance between endocrine and exocrine cell differentiation.