The platypus (Ornithorhynchus anatinus), a monotreme, is an evolutionary conundrum, seemingly composed of both ancestral reptilian and derived mammalian traits. Like reptiles and birds, the female platypus lays eggs, but after hatching the young are fed with milk, as is the case with all mammals. Its genome is similarly an amalgam of reptilian and mammalian features. Monotremes diverged from therian mammals (the line that gave rise to marsupials and eutherians) approximately 166 million years ago. Thus, the analysis of pluripotent stem cells from the platypus should provide unique insights into the evolution of pluripotency in mammals. We have generated the first pluripotent stem cells from a monotreme, the platypus, by reprogramming dermal fibroblasts from an adult female platypus into induced pluripotent stem cells (piPSCs). Platypus iPSCs are LIF-dependent, show alkaline phosphatase activity and express the cell surface markers SSEA1, SSEA4, TRA1-60 and TRA1-81. In vitro, piPSC are able to give rise to derivatives of all three germ layers. Deep sequencing of the piPSC transcriptome revealed that the eutherian core pluripotency factors OCT4, SOX2 and NANOG are ancestral regulators of pluripotency, while other eutherian pluripotency factors, such as ESRRB and NR0B1/DAX1, have been more recently co-opted into the pluripotency network as they are not expressed in piPSCs. Analysis of the DAX1 promoter reveals the absence of a SOX2 DNA binding site known to be critical for DAX1 expression in eutherian pluripotent stem cells. Similarly, the platypus ESRRB gene lacks two SOX2 binding sites within intron 2 which are conserved amongst eutherians. The recruitment of ESRRB and DAX1 into the pluripotency regulatory network during eutherian evolution thus seems to be due to the acquisition of SOX2 responsiveness. Platypus iPSCs allow the first glimpse into the early development of a monotreme in addition to providing novel insights into the evolution of mammalian pluripotency.