The Notch pathway is involved in cell differentiation processes in various organs and at several developmental stages. The importance of Notch for early T lymphocyte development is well established. Recently, Notch has been implicated in directing naive T helper cell differentiation towards the Th1, Th2 or regulatory T cell lineages. However, the molecular events underlying these processes are poorly understood. We show that the Notch ligands Delta-like1, Delta-like4 and Jagged1 differentially affect early T cell activation and proliferation following T cell receptor cross-linking. Delta-like1 and Jagged1 induce a dose-dependent inhibition of early activation markers CD69 and CD25, as well as inhibition of proliferation after anti-CD3 stimulation of purified CD4+ T cells. Similarly, the rapid activation of transcription factors NF-AT, AP-1 and NF-kappaB is suppressed. In contrast, triggering of Notch by Delta-like4 enhances T cell activation and proliferation. The observed effects are dependent on simultaneous cross-linking of TCR and Notch but independent of gamma-secretase-mediated cleavage of Notch. These data suggest direct interference between Notch and early TCR signal transduction events, independent of the classical Notch pathway via release of the Notch intracellular domain. A Notch-mediated alteration of TCR signaling strength may contribute to the recently described modulation of naïve T cell differentiation by Notch ligands.