Notch and HOXB4 have been reported to expand hematopoietic stem cells (HSCs) in vitro. However, their critical effector molecules remain undetermined. We found that the expression of c-myc, cyclin D2, cyclin D3, cyclin E, and E2F1 was induced or enhanced during Notch1- or HOXB4-induced self-renewal of murine HSCs. Since c-Myc can act as a primary regulator of G(1)/S transition, we examined whether c-Myc alone can induce self-renewal of HSCs. In culture with stem cell factor, FLT3 ligand, and IL-6, a 4-hydroxytamoxifen-inducible form of c-Myc (Myc/ERT) enabled murine Lin(-)Sca-1(+) HSCs to proliferate with the surface phenotype compatible with HSCs for more than 28 days. c-Myc activated by 4-hydroxytamoxifen augmented telomerase activities and increased the number of CFU-Mix about 2-fold in colony assays. Also, in reconstitution assays, HSCs expanded by c-Myc could reconstitute hematopoiesis for more than 6 months. As for the mechanism of c-myc induction by Notch1, we found that activated forms of Notch1 (NotchIC) and its downstream effector recombination signal-binding protein-J kappa (RBP-VP16) can activate the c-myc promoter through the element between -195 bp and -161 bp by inducing the DNA-binding complex. Together, these results suggest that c-Myc can support self-renewal of HSCs as a downstream mediator of Notch and HOXB4.