Hematopoiesis in the mouse conceptus begins in the visceral yolk (VYS), with primitive erythroblasts first evident in blood islands at the headfold stage (E8.0). VYS erythropoiesis is decreased or abrogated by targeted disruption of the hematopoietic transcription factors tal-1, rbtn2, GATA-1, and GATA-2. To better understand the potential roles of these genes, and to trace the initial temporal and spatial development of mammalian embryonic hematopoiesis, we examined their expression patterns, and that of betaH1-globin, in normal mouse conceptuses by means of in situ hybridization. Attention was focused on the 36-hour period from mid-primitive streak to early somite stages (E7.25 to E8.5), when the conceptus undergoes rapid morphologic changes with formation of the yolk sac and blood islands. Each of these genes was expressed in extraembryonic mesoderm, from which blood islands are derived. This VYS expression occurred in a defined temporal sequence: tal-1 and rbtn2 transcripts were detected earlier than the others, followed by GATA-2 and GATA-1, and then by betaH1-globin. Transcripts for all of these genes were present in VYS mesoderm cell masses at the neural plate stage (E7.5), indicating commitment of these cells to the erythroid lineage before the appearance of morphologically recognizable erythroblasts. By early somite stages (E8.5), GATA-2 mRNA expression is downregulated in VYS blood islands as terminal primitive erythroid differentiation proceeds. We conclude that primitive mammalian erythropoiesis arises during gastrulation through the ordered temporal expression of tal-1, rbtn2, GATA2, and GATA-1 in a subset of extraembryonic mesoderm cells. During the stages analyzed, tal-1 and rbtn2 expression was also present in posterior embryonic mesoderm, while GATA-1 and GATA-2 expression was evident in extraembryonic tissues of ectodermal origin.