Notch receptors are involved in lineage decisions in multiple developmental scenarios, including hematopoiesis. Here, we treated hybrid human-mouse fetal thymus organ culture with the gamma-secretase inhibitor 7 (N-[N-(3,5-difluorophenyl)-l-alanyl]-S-phenyl-glycine t-butyl ester) (DAPT) to establish the role of Notch signaling in human hematopoietic lineage decisions. The effect of inhibition of Notch signaling was studied starting from cord blood CD34(+) or thymic CD34(+)CD1(-), CD34(+)CD1(+), or CD4ISP progenitors. Treatment of cord blood CD34(+) cells with low DAPT concentrations results in aberrant CD4ISP and CD4/CD8 double-positive (DP) thymocytes, which are negative for intracellular T-cell receptor beta (TCRbeta). On culture with intermediate and high DAPT concentrations, thymic CD34(+)CD1(-) cells still generate aberrant intracellular TCRbeta(-) DP cells that have undergone DJ but not VDJ recombination. Inhibition of Notch signaling shifts differentiation into non-T cells in a thymic microenvironment, depending on the starting progenitor cells: thymic CD34(+)CD1(+) cells do not generate non-T cells, thymic CD34(+)CD1(-) cells generate NK cells and monocytic/dendritic cells, and cord blood CD34(+)Lin(-) cells generate B, NK, and monocytic/dendritic cells in the presence of DAPT. Our data indicate that Notch signaling is crucial to direct human progenitor cells into the T-cell lineage, whereas it has a negative impact on B, NK, and monocytic/dendritic cell generation in a dose-dependent fashion.