We are interested in exploiting the mechanisms that control self- renewal and differentiation of mouse embryonic stem cells. These cells originate from the Inner Cell Mass (ICM) of the blastocyst and give rise to all tissues of the embryo including the germline. ES cells are a powerful experimental system to understand the mechanisms that underlie developmental processes.rnrnEmbryonic stem cells can differentiate into all 3 germ layers in culture. During differentiation the pluripotent ES cells give rise to progenitor cells that in their turn give rise to terminally differentiated cells. The process of differentiation is guided by the various factors that are added to the culture media and is not yet well understood. It is important to understand this process in order to guide and control it. For this reason we have developed (and continue to develop) various genetic experimental tools. One approach is to conditionally immortalize using tetracycline controlled SV40 large T-Antigen the various cell types that arise during ES cell differentiation. The expression of Large T-Antigen is induced during differentiation followed by cellular cloning. During the induction period the cells proliferate. After ending the induction the cells can be terminally differentiated and characterized with the help of existing markers. The goal is to expand homogeneous populations of cells that undergo differentiation and compare their expression profile and epigenetic status.rnrnIn other words, we have developed a system that enables conditional immortalization of cell populations that arise during ES cell differentiation. This technical advance opens a door to new mechanistic studies on gene expression hierarchies in differentiation.