The mammalian preimplantation embryo comprises three distinct lineages; trophectoderm, primitive endoderm and epiblast. The epiblast lineage is the founder line of all the embryonic tissues, while the other lineages give rise to extra-embryonic tissues. The mechanisms behind formation and specification of the first lineages are not fully understood. Pluripotency is a unique characteristic of epiblast lineage. SOX2 and KLF4 transcription factors are a part of the core pluripotency networks, and are known to play a role in somatic cells reprogramming. The role of both factors has been extensively studied in vitro; however, their role in the establishment of pluripotency in vivo is not well understood. This thesis investigated the role of SOX2 and KLF4 in the formation and specification of epiblast lineage in preimplantation mammalian embryo. For many technical reasons, mouse embryo was used as a model in this study. Examination of SOX2 and KLF4 localisation during preimplantation development by immunochemistry revealed that although both proteins were previously proposed to be important components of pluripotency circuit, only SOX2 localisation was restricted to the pluripotent lineages, whereas KLF4 expression was broader and did not mark the pluripotent line throughout the whole preimplantation period. SOX2 levels and localisation were controlled by FGF4/Erk pathway, whereas KLF4 was not. To analyse the function of SOX2 and KLF4, eight-cell stage embryos were injected with Sox2 and Klf4 mRNAs and double-stranded RNAs, and were cultured for 48-72 hours to follow up the progeny of the injected cells. Functional analysis revealed that SOX2 and KLF4 together are crucial to the formation and specification of epiblast lineage. In conclusion, for successful specification of pluripotent line in vivo two mechanisms are needed, first the up-regulation of the pluripotency factors (mediated by SOX2 and KLF4), second simultaneous down-regulation of key endoderm-differentiation factor GATA6 (mediated by KLF4).