During early embryogenesis, the transcription factor HNF3β is expressed in visceral and definitive endoderm, node, notochord and floorplate. A targeted mutation in the HNF3β gene results in the lack of a definitive node and notochord. Furthermore, lack of HNF3β results in failure of proper primitive streak elongation. To address whether HNF3β is required in visceral endoderm, we have used tetraploid embryo-ES cell aggregations to generate chimeric mouse embryos with wild-type visceral endoderm and homozygous mutant HNF3β embryonic ectoderm or vice versa. Replacing the visceral endoderm of mutant HNF3β embryos rescued proper primitive streak elongation and, conversely, mutant visceral endoderm imposed a severe embryonic-extraembryonic constriction on wild-type embryonic ectoderm. Restoration of normal streak morphogenesis was not sufficient to allow formation of the node and notochord in HNF3β mutant embryos. Thus, our results demonstrate that HNF3β has two separate roles in primitive streak formation. One is to act within the visceral endoderm to promote proper streak morphogenesis. The second is autonomous to the node and its precursors and involves specification of node and notochord cell fates. HNF3β mutant embryos rescued for the embryonic-extraembryonic constriction developed further than mutant embryos, allowing examination of later roles for HNF3β. We show that such mutant embryos lack foregut and midgut endoderm. In addition, left-right asymmetry is affected in the mutant embryos.