Klf5 functions as a multi-lineage reprogramming transcription factor

The transcription factor Klf5 is indispensable for both embryonic (redundantly with Klf4) and extra-embryonic development during pre-implantation, yet its role in re-establishing these different fates in vitro remains largely unexplored. Here, we demonstrate that in the absence of WNT signaling activation at the beginning, ectopic expression of Klf5 in post-implantation epiblast stem cells (EpiSCs) not only induces naïve pluripotency, but also exclusively drives reprogramming into trophoblast stem cells (TSCs) and primordial germ cells (PGCs) by directly reactivating key lineage-specific determinants. Single-cell transcriptome analysis reveals distinct reprogramming trajectories toward these three fates, alongside a non-reprogramming trajectory that driven by Klf5-mediated BMP signaling activation. Moreover, endogenous Klf5 is crucial for establishing TSC and PGC fates from embryonic stem cells driven by other inducers. Our findings highlight Klf5’s multipotent reprogramming capacity and its pivotal role in early TSC and PGC fate specification, offering new insights into its versatile functions during embryonic development.