Initial embryological development in avian species, consisting of cleavage and area pellucida formation, occurs prior to oviposition. In chickens, the first lineage segregation is known to occur during the last 10 hours of intrauterine development, a finding which has primarily been identified on the basis of morphological perspectives. We traced the early expression of the transcription factors NANOG, POUV and EOMES at Eyal-Giladi and Kochav (EGK) stages VI through X using in situ hybridization. At EGK.VI, NANOG and EOMES were heterogeneously expressed in a salt-and-pepper manner. From EGK.VIII to EGK.X, NANOG- or EOMES-positive cells were predominantly located in the epiblast or area opaca regions, respectively. POUV-expressing cells were found in the upper layer at EGK.VIII. After oviposition, POUV mRNA was strongly expressed in the epiblast, but weakly expressed in the hypoblast at EGK.X. Furthermore, NANOG- and POUV-negative cells were located in the subgerminal cavity where layer reduction occurs during area pellucida formation. Those cells were larger and did not seem to contribute to epithelialization until EGK.X. Our results on the spatiotemporal expression of transcription factors contribute to a greater understanding of the dynamic process of intrauterine development in chickens.