Within the process of lymphocyte differentiation, activation of the Notch signaling pathway specifies the T cell lineage and disallows development along the default B cell pathway. Notch signaling is tightly controlled, and regulators of the Notch pathway exert dramatic effects on the T/B lineage decision in developing lymphocytes. The E3 ubiquitin ligase Itch has been shown to bind and ubiqutinate Notch1, but the consequences of this interaction have not been described. We demonstrate that Itch is expressed in fetal liver hematopoietic progenitor cells and thymocytes and upregulated in response to Notch signaling. Overexpression of Itch in hematopoietic progenitor cells co-cultured with OP9-DL1 stromal cells subtly inhibits T cell development and promotes B cell development suggesting inhibition of Notch signaling. Itch overexpression appears to diminish the rate of Notch1 degradation. These results suggest a role for the ubiquitin ligase Itch in antagonizing Notch signaling during lymphocyte development through a non-degradative mechanism.