We generated a CRISPR/Cas9 mediated knockout (KO) of the OCT4 gene in adult fibroblasts, where biallelic deletion of a single nucleotide leads to a frameshift mutation. Through reconstruction of embryos by somatic cell NT, we were able to study the role of OCT4 during pre-implantation development until Day 7 in vitro. The presence of OCT4 protein was evaluated after immunofluorescent staining by confocal laser scanning microscopy of Day 5 morulae and Day 7 blastocysts; somatic cell NT embryos reconstructed from nontransfected cells of the same source served as control. Whereas control morulae expressed OCT4 in all cells, OCT4 KO morulae showed expression in only 67.8 ± 11.1% (mean ± SD, n = 6) of cells and overall intensity was decreased. By Day 7, no expression of OCT4 was detected in OCT4 KO blastocysts (n = 24), suggesting that maternal stores of the OCT4 protein had decayed. In contrast, control blastocysts (n = 20) showed OCT4 expression ubiquitously in both inner cell mass (ICM) and trophectoderm (TE). Simultaneously to the OCT4 staining, we differentially stained ICM and TE with the TE specific marker CDX2 and counterstained cell nuclei with 4′,6-diamidino-2-phenylindole. No significant differences between OCT4 KO Day 7 blastocysts and controls were detected in total cell numbers (89.6 ± 27.5 v. 96.3 ± 38) and percentage of CDX2 positive cells (50.7 ± 16.8% v. 59.0 ± 20.8%) (P > 0.05, mean ± SD, unpaired, two-tailed t-test). To analyse the role of OCT4 during the second lineage differentiation, we stained Day 5 morulae and Day 7 blastocysts for the epiblast and hypoblast specific markers NANOG and GATA6, respectively. In morulae, both markers were present and co-expressed in OCT4 KO and control embryos. By Day 7, control blastocysts (n = 6) already showed the typical salt and pepper distribution of NANOG and GATA6 positive cells, but expression was not mutually exclusive in all cells and also not restricted to ICM. OCT4 KO embryos lost all NANOG expression at Day 7 blastocyst stage (n = 8) and only stained positive for GATA6 in both TE and ICM. We conclude that OCT4 is not required for the quantitative allocation of cells to either the ICM or the TE during the first lineage differentiation, as total cell number and percentage of CDX2 positive cells was unchanged. Additionally, expression of NANOG seems to be OCT4 dependent and OCT4 KO embryos fail to establish the epiblast lineage—unlike mouse Oct4 KO embryos, where developmental failure was connected to loss of GATA6 expression during second lineage differentiation.