Understanding the intrinsic and extrinsic signals that regulate the molecular basis of the pluripotent state may improve our understanding of mammalian embryogenesis, different states of pluripotency, and our ability to tailor lineage differentiation. Although the role of the PI3K/Akt pathway in the self-renewal and maintenance of mESCs is well-established, the specific contribution of the pathway or of its negative regulator, PTEN, in the maintenance of the human pluripotent state is less understood. To explore the PI3K/AKT pathway in human embryonic stem cell (hESC) pluripotency and differentiation, we generated stable PTEN knockdown (KD) hESCs using short hairpin RNA. Similar to mESCs, we found that PTEN KD hESCs have increased self-renewal, cell survival, and proliferation over multiple passages compared to control cells. However, in contrast to mESCs, in vitro, PTEN KD hESCs differentiated inefficiently in directed differentiation assays, in part due to the continued maintenance of OCT4 and NANOG expression. In teratoma assays, PTEN KD hESCs generated tissues from the three germ layers, although with a bias toward neuroectoderm differentiation. These results demonstrate that PTEN is a key regulator of hESC growth and differentiation, and manipulation of this pathway may improve our ability to regulate and understand the pluripotent state. STEM CELLS 2011;29:1952–1962.