Differentiation procedures leading to osteogenic and chondrogenic differentiation of embryonic stem cells (ESCs) have been established and well upgraded over the past decade. Novel cell-culture conditions, signaling inducers, and chemical modifications of cellular environment have been found and optimized for use as steering or supporting modules in ESC differentiation. While most of the novel studies of osteoblasts or chondrocytes differentiated from ESCs deal with their regenerative potential, the “childhood diseases” of basic differentiation have not yet been quite solved. Purification procedures are still facing a lack of exclusive markers for osteogenic progenitors and a collateral development of other cell types at the end points of differentiation that possibly lead to teratomas. This chapter discusses the role of novel markers and inducers in osteogenic and chondrogenic differentiation, their effect on signaling pathways, particularly on that of Wnt/beta-catenin, and the time-specific manner of their action. We present an improved osteogenic differentiation protocol based on the hanging drop method and a time-optimized use of 1α,25-(OH)2 vitamin D3 , all-trans retinoic acid, and bone morphogenetic protein 2 (BMP-2) with an end point efficiency increased up to 90% and a protocol for chondrogenic differentiation, which employs BMP-2 and transforming growth factor β1 as chondrogenic inducers, with 60% chondrogenic end point efficiency.
