Human pluripotent stem cells (hPSCs), including human embryonic stem cells and induced pluripotent stem cells, are a potentially inexhaustible supply of human cells because they can be propagated indefinitely while still retaining the capacity to differentiate into all somatic cell types. Hurdles facing utilization of hPSCs in regenerative medicine include lack of reliable and efficient methods to differentiate hPSCs into disease related cell lineages, including neurons, cardiac muscle cells and pancreatic beta cells. During differentiation, hPSCs make a number of developmental decisions to generate first the three germ layers (ectoderm, mesoderm and endoderm), followed by subsequent decisions to generate all of the body's mature tissues. These differentiation processes often involve a limited set of developmental cell signaling pathways, which can be modulated by growth factors and small molecules. Therefore, common methods of differentiating hPSCs to therapeutic applicable cell lineages require application of specific combination of growth factors and/or small molecules at distinct differentiation stages. This chapter discusses recent progress in the production of therapeutically applicable cells via directed differentiation of hPSCs.