There is growing evidence that thymic epithelial cells (TEC) are the key cell type responsible for orchestrating the fetal development and postnatal function. A single transcription factor, FOXN1, is known to be a critical regulator of multiple aspects of TEC proliferation and function throughout the lifespan. The TEC compartment switches from a fetal/neonatal expansion program to a juvenile homeostasis program, with a key transition point occurring at about 7 days postnatal in mice (P07), and about 4 months of age in humans. Data in mice show that this switch is controlled by the retinoblastoma (RB) pathway, acting at least in part by suppression of Foxn1 gene expression by E2F transcription factors. Mice deficient for multiple RB family members fail to make this switch and continue to expand. The K5.D1 transgenic line, in which CyclinD1 is specifically overexpressed in TEC, mimics this RB loss of function phenotype by activating cyclin-dependent kinases that inhibit RB function. In both RB mutants and K5.D1 transgenics Foxn1 gene expression is elevated, and suppressing Foxn1 expression using a postnatal-specific hypomorphic allele (Foxn1Z/Z) restores the fetal to juvenile switch, normalizing thymus size. We have also shown that Foxn1 regulation of TEC proliferation occurs primarily in MHCIIlo TEC, consistent with preliminary data from the Richie lab that a Sca1-MHCIIlo TEC subset may contain a key proliferating progenitor population during the switch from fetal expansion to juvenile homeostasis. These data suggest that during the perinatal to juvenile transition, RB proteins modulate Foxn1 transcription via E2F transcription factors differentially in specific TEC subsets to ?put the brakes on? TEC proliferation, and thus regulate organ size. There is also growing evidence that the neonatal and adult thymi have stage-specific functions that generate distinct T cell populations. As FOXN1 is also known to be a key regulator of TEC differentiation, the changes in how Foxn1 expression is regulated to control TEC proliferation should also have significant impacts on TEC function in the neonatal and adult thymus. We propose three specific aims to test the hypothesis that the RB-dependent changes in Foxn1 gene expression levels during the neonatal period that are essential for establishing organ homeostasis are also required to generate specific microenvironments that are necessary for neonatal-specific organ functions. The goals of this project are to determine: how changes in Foxn1 expression across this transition impact stromal composition changes in mouse, and compare to stromal changes in human thymus; how Foxn1 expression is regulated by the RB pathway during this transition; and how these Foxn1-dependent processes impact immune function.