Factors that trigger and sustain self-renewal divisions in tissue stem cells remain poorly characterized. By modulating the levels of Hoxb4 and its co-factor Pbxl in primary hematopoietic cells (Hoxb4hiPbxl(10) cells), we report an in vitro expansion of mouse hematopoietic stem cells (HSCs) by 105-fold over 2 weeks, with subsequent preservation of HSC properties. Clonal analyses of the hematopoietic system in recipients of expanded HSCs indicate that up to 70% of Hoxb4hiPbxl(10) stem cells present at initiation of culture underwent self-renewal in vitro. In this setting, Hoxb4 and its co-factor did not promote an increase in DNA synthesis, or a decrease in doubling time of Scal+Lin- cells when compared to controls. Q-PCR analyses further revealed a downregulation of Cdknlb (p27Kipl) and Mxdl (MadI) transcript levels in Hoxb4hiPbxl(l0) primitive cells, accompanied by a more subtle increase in c-myc and reduction in Ccnd3 (Cyclin D3). We thus put forward this strategy as an efficient in vitro HSC expansion tool, enabling a further step into the avenue of self-renewal molecular effectors.