2It is well recognized that thymocytes must undergo a progression of maturation and differentiation stages on their journey to becoming a mature CD4+ or CD8+ T cell. Early is this progression are four double-negative (DN) stages, so named because they express neither CD4 or CD8 on their surface. These DN stages have been further classified as DN1, DN2, DN3, or DN4 based on the presence or absence of the specific cell membrane markers, CD25 and CD44. The process of a cell progressing from one DN stage to another (and beyond) is a complex “choreography” involving the action of numerous transcription factors, morphogens, and cytokines. The first critical component in this sequence is the activation of the transmembrane receptor Notch1. Failure to engage the Notch1 receptor at this stage shunts the cells into an entirely different pathway that results in production of NK cells, B cells, or myeloid cells. However, if the cell passes this critical step, it then begins the process of becoming a mature T cell by the sequential action of the aforementioned transcription factors, morphogens, and cytokines. In this chapter we discuss how these factors contribute to the maturation of T cells by detailing the consequences of the loss of the action of each of the factors involved in DN maturation.