Recent advances regarding the biology of adipose tissue have demonstrated that white adipose tissue (WAT) plays a central role in the regulation of energy balance and acts as a secretory/endocrine organ that mediates numerous physiological and pathological processes. Dysregulation of WAT mass causes obesity or lipoatrophy, two disorders associated with life-threatening pathologies, including cardiovascular diseases and diabetes. Alterations in WAT mass result from changes in adipocyte size and/or number. Change in adipocyte number is achieved through a complex interplay between proliferation and differentiation of preadipocytes. Adipocyte differentiation or adipogenesis is a highly controlled process that has been extensively studied for the last 25 years. In vitro preadipocyte culture systems that recapitulate most of the critical aspects of fat cell formation in vivo have allowed a meticulous dissection of the cellular and molecular events involved in the adipogenesis process. The adipogenic transcription factors peroxisome proliferator-activated receptor-gamma and CCAAT/enhancer binding protein-alpha play a key role in the complex transcriptional cascade that occurs during adipogenesis. Hormonal and nutritional signaling affects adipocyte differentiation in a positive or negative manner, and components involved in cell-cell or cell-matrix interactions are also pivotal in regulating the differentiation process. This knowledge provides a basis for understanding the physiological and pathophysiological mechanisms that underlie adipose tissue formation and for the development of novel and sound therapeutic approaches to treat obesity and its related diseases.