Differentiation of CD4+ helper and CD8+ cytotoxic αβ T cells from CD4+CD8+ thymocytes involves up-regulation of lineage-specifying transcription factors and transcriptional silencing of CD8 or CD4 co-receptors respectively in major histocompatibility complex (MHC) II or I restricted thymocytes. inactivation of the p53 pro-apoptotic protein rescues these thymocytes from apoptosis increasing their frequency and permitting accumulation of CD4+CD8+ αβ T cells in the periphery. Dicer-deficient MHCI-restricted αβ T cells fail to normally silence and display impaired induction of the CD8-lineage specifying transcription factor Runx3 whereas Dicer-deficient MHCII-restricted αβ T cells show impaired silencing and impaired induction of the CD4-lineage specifying transcription factor Thpok. Finally we show that this Drosha RNA endonuclease which functions upstream of Dicer in microRNA SB939 biogenesis also regulates and silencing. Our data demonstrate a previously dismissed function for the microRNA biogenesis machinery in regulating expression of lineage-specifying transcription factors and silencing of and during αβ T cell differentiation. Introduction The generation of distinct cellular lineages from multipotent progenitor cells involves differentiation programs that couple up-regulation of lineage specific genes with silencing of genes expressed in progenitor cells and option lineages. The initiation maintenance and silencing of gene expression during lineage commitment are regulated by genetic and epigenetic mechanisms. One paradigm for elucidating molecular mechanisms that control gene expression during lineage commitment is the differentiation of CD4+ and CD8+ αβ T cells from CD4+CD8+ (double-positive or DP) thymocytes that have expressed functional αβ TCRs (1 2 Assembly and expression of T cell receptor (TCR) β genes drives CD4?CD8? (double-negative or DN) thymocytes to differentiate into DP thymocytes (3). This developmental transition initiates rearrangement and expression of TCRα genes which leads to expression of unique αβ TCRs on immature CD4+CD8+ thymocytes. Specificities of αβ TCRs are selected through interactions of these antigen receptors with self-peptide/MHC complexes expressed on thymic epithelial cells a process aided by CD4 and CD8 co-receptors (3 4 Depending on the affinity of such interactions thymocytes die by “neglect” are rescued NT5E from programmed cell death and further differentiate (positive selection) or are actively deleted (unfavorable selection). Concomitant with positive selection immature CD4+CD8+ thymocytes up-regulate lineage-specifying transcription factors and silence or as they differentiate into mature CD4+ or CD8+ (single positive or SP) thymocytes. SP cells exit the thymus and migrate to the spleen lymph nodes and other peripheral organs as CD4+ or CD8+ lineage αβ T cells. Differentiation of CD4+ and CD8+ αβ T cells is usually regulated by αβ TCR-activated signaling pathways that control downstream transcription factors (2 5 These factors include Runx3 which is required for CD8 lineage effector functions and silencing and Thpok (encoded by silencing (2 6 Runx3 and Thpok are mutually antagonistic as Runx3 represses expression by binding a silencer upstream of the promoter (11 12 while Thpok represses expression (13-15) and antagonizes Runx-mediated repression of silencer (14 16 Despite requirement for Runx3 and the silencer in initiation of silencing neither is required to prevent re-expression in peripheral CD8+ αβ T cells (17 18 implying that silencing SB939 is usually maintained epigenetically. In contrast to control of expression lineage-specific transcription appears SB939 to be regulated by developmental stage specific enhancers rather than a enhancers may facilitate silencing SB939 in CD4+ cells (10). In addition to Runx3 and Thpok several transcription factors and chromatin modifying enzymes modulate CD4/CD8 lineage commitment and/or co-receptor expression yet none of these has been shown to directly regulate initiation of or silencing following positive selection of DP thymocytes (1 2 23 The Dicer and Drosha RNA endonucleases guideline cellular differentiation through their ability to control gene expression. Both proteins are required for the biogenesis of microRNAs (miRs) which repress gene expression by binding and destabilizing or blocking translation of mRNAs (24). However Dicer can also function independently of Drosha to.