Osteogenic differentiation is a tightly regulated process realized by progenitor cell osteoblasts. Notch signaling pathway plays a critical role in skeletal development and bone remodeling. Controversial data exist regarding the role of Notch activation in promoting or preventing osteogenic differentiation. This study aims to investigate the effect of several Notch components and their dosage on osteogenic differentiation of mesenchymal stem cells of adipose tissue. Osteogenic differentiation was induced in the presence of either of Notch components (NICD, Jag1, Dll1, Dll4) dosed by lentiviral transduction. We show that osteogenic differentiation was increased by NICD and Jag1 transduction in a dose-dependent manner; however, a high dosage of both NICD and Jag1 decreased the efficiency of osteogenic differentiation. NICD dose-dependently increased activity of the CSL luciferase reporter but a high dosage of NICD caused a decrease in the activity of the reporter. A high dosage of both Notch components NICD and Jag1 induced apoptosis. In co-culture experiments where only half of the cells were transduced with either NICD or Jag1, only NICD increased osteogenic differentiation according to the dosage, while Jag1-transduced cells differentiated almost equally independently on dosage. In conclusion, activation of Notch promotes osteogenic differentiation in a tissue-specific dose-dependent manner; both NICD and Jag1 are able to increase osteogenic potential but at moderate doses only and a high dosage of Notch activation is detrimental to osteogenic differentiation. This result might be especially important when considering possibilities of using Notch activation to promote osteogenesis in clinical applications to bone repair.