In recent years, there has been a great deal of interest in the development of regenerative approaches to produce hyaline cartilage ex vivo that can be utilized for the repair or replacement of damaged or diseased tissue. It is clinically imperative that cartilage engineered in vitro mimics the molecular composition and organization of and exhibits biomechanical properties similar to persistent hyaline cartilage in vivo. Experimentally, much of our current knowledge pertaining to the regulation of cartilage formation, or chondrogenesis, has been acquired in vitro utilizing high-density cultures of undifferentiated chondroprogenitor cells stimulated to differentiate into chondrocytes. In this review, we describe the extracellular matrix molecules, nuclear transcription factors, cytoplasmic protein kinases, cytoskeletal components, and plasma membrane receptors that characterize cells undergoing chondrogenesis in vitro and regulate the progression of these cells through the chondrogenic differentiation program. We also provide an extensive list of growth factors and other extracellular signaling molecules, as well as chromatin remodeling proteins such as histone deacetylases, known to regulate chondrogenic differentiation in culture. In addition, we selectively highlight experiments that demonstrate how an understanding of normal hyaline cartilage formation can lead to the development of novel cartilage tissue engineering strategies. Finally, we present directions for future studies that may yield information applicable to the in vitro generation of hyaline cartilage that more closely resembles native tissue.