The genetic mechanisms that promote lineage commitment and eliminate autoreactive cells in the thymus are not well understood. To better understand this process, we have identified and quantitated transcripts in the two major thymocyte lineages by using serial analysis of gene expression. Approximately 25 genes displayed almost complete segregation to one or the other T cell lineage. Commitment to the CD4 lineage was marked by up-regulation of genes associated with increased survival and chaperone function followed by expression of genes that regulate nucleosome remodeling and T cell receptor signaling. Differentiation within the CD8 lineage, on the other hand, was marked by up-regulation of genes that regulate lymphocyte homing, followed by quenching of genes that inhibit apoptosis. Definition of differential gene expression during development of the two major thymocyte lineages will allow insight into mechanisms of T cell development after positive and negative selection.