Human embryonic stem (hES) cells can self-renew in culture and differentiate into cells of all three germ layers. Identification of regulators of the self-renewal versus differentiation decisions in hES cells will facilitate manipulation of these cells. Several lines of evidence suggest that CITED2 might play a role in regulation of stem cell functions. To test this, forced expression of CITED2 was peformed by transducing hES cells with a virus containing a CITED2-IRES-GFP cassette. CITED2 overexpression was confirmed at a RNA and protein level. Analysis of cell morphology, expression of pluripotency factors and cell surface markers, as well as the ability to differentiate, suggest that CITED2-overexpressing cells remain in an undifferentiated pluripotential state. However, these cells possess a 2-fold higher clonogenic efficiency and produce larger colonies than control cells. In order to test whether CITED2 overexpression could maintain properties of undifferentiated hES cells in the absence of bFGF, CITED2-overexpressing cells were cultured with and without bFGF, which according to several studies is required for hES cell self-renewal. In contrast to control cells, which do not survive without bFGF, CITED2-overexpressing cells can be maintained in culture for several passages. In addition, CITED2 was knocked down in hES cells. Preliminary results show that the cells die suggesting that CITED2 is required for maintenance of self-renewing hES cell cultures. While these results point to a role for CITED2 in hES cell self-renewal, it is interesting to note that as judged by global gene expression profiling and qPCR analysis, CITED2 is more strongly expressed in differentiated subcompartments of hES cells. Further experiments are required to determine the exact mechanism by which CITED2 acts in hES cells but forced expression of CITED2 might alter the probability of self-renewal versus differentiation. This may be effected in part through the bFGF pathway.