Human artificial chromosomes (HACs), including the de novo synthesized alphoidtetO-HAC, are a powerful tool for introducing genes of interest into eukaryotic cells. HACs are mitotically stable, non-integrative episomal units that have a large transgene insertion capacity and allow efficient and stable transgene expression. Previously, we have shown that the alphoidtetO-HAC vector does not interfere with the pluripotent state and provides stable transgene expression in human induced pluripotent cells (iPSCs) and mouse embryonic stem cells (ESCs). In this study, we have elaborated on a mouse model of ex vivo iPSC- and HAC-based treatment of hemophilia A monogenic disease. iPSCs were developed from FVIIIY/− mutant mice fibroblasts and FVIII cDNA, driven by a ubiquitous promoter, was introduced into the alphoidtetO-HAC in hamster CHO cells. Subsequently, the therapeutic alphoidtetO-HAC-FVIII was transferred into the FVIIIY/– iPSCs via the retro-microcell-mediated chromosome transfer method. The therapeutic HAC was maintained as an episomal non-integrative vector in the mouse iPSCs, showing a constitutive FVIII expression. This study is the first step towards treatment development for hemophilia A monogenic disease with the use of a new generation of the synthetic chromosome vector—the alphoidtetO-HAC.