The development of efficient, reproducible protocols for directed in vitro differentiation of hES cells into insulin producing beta cells will benefit greatly from increased knowledge regarding the spatiotemporal expression profile of key instructive factors involved in human endocrine cell generation. Human fetal pancreases, from 7 to 21 weeks of gestational age, were collected following consent immediately after pregnancy termination and processed for immunostaining, in situ hybridization and real-time RT-PCR expression analyses. Islet-like structures appear from approximately week 12 and unlike the mixed architecture observed in the adult islets, fetal islets are initially formed predominantly by aggregated insulin or glucagon-expressing cells. The period studied (7-22 weeks) coincides with a decrease in the proliferation and an increase in the differentiation of the progenitor cells, the initiation of NGN3 expression and the appearance of differentiated endocrine cells. The present study provides a detailed characterization of islet formation and expression profiles of key intrinsic and extrinsic factors during human pancreas development. This information is beneficial for the development of efficient protocols that will allow guided in vitro differentiation of hES cells into insulin-producing cells.