Human embryonic stem cells (hESCs) have the potential to provide alternative sources for pancreatic islet grafts. In the present study we have investigated the influence of Activin A and Activin B on the expression of the pancreas marker gene PdV1 in hESCs differentiated as embryoid bodies (EBs). We report here that Activin B in a dose depend manner markedly up-regulates Pdx1 expression as compared to Activin A and untreated cultures. Pdv1(+) cells co-express FOXA2 but lacks, however, co-expression with nkV6.1, a marker combination that in the present study is shown precisely to identify embryonic and fetal pancreas antage in humans. Pdx1(+) cells are found in cell clusters also expressing Serpinal and FABP1, suggesting activation of intestinal/liver developmental programs. Moreover, Activin B up-regulates Sonic Hedgehog (Shh) and its target Glil, which during normal development is suppressed in the pancreatic anlage. In conclusion, Activin B is a potent inducer of PdY1 as well as Shh in differentiating hESCs. The data suggest that additional suppression of Shh signaling may be required to allow for proper specification of pancreatic cell lineages in hESCs. (c) 2007 Elsevier Inc. All rights reserved.