The use of human pluripotent stem cells (hPSCs) is hampered by tumor forming potential and limited ability to generate pure populations of differentiated cell types in vitro. To address these issues, it is desirable to generate more mature, self-renewable, intermediate stem cell populations from hPSCs. We established Endodermal Stem (EP) cell lines from hPSCs, which have unlimited self-renewal and are non-tumorigenic. Clonally derived EP cells differentiate into numerous endodermal lineages in vivo and in vitro, including mono-hormonal functional β-cells, hepatocytes and intestinal epithelia. EP cells represent a powerful tool to study endoderm specification and offer a safer source of endodermal derived tissues for transplantation therapies.