Ectopic expression of the homeodomain transcription factor HOXB4 expands hematopoietic stem and progenitor cells in vivo and in vitro, making HOXB4 a highly interesting candidate for therapeutic stem cell expansion. However, when expressed at high levels, HOXB4 concomitantly perturbs differentiation and thus likely predisposes the manipulated cells for leukemogenesis. We therefore asked whether the expression level of HOXB4 may be a critical parameter that influences the growth and transformation properties of transduced cells. Using a set of retroviral vectors which covered a 40-fold range of expression levels, we studied the consequences of HOXB4 expression at different levels in the well established Rat-1 fibroblast cell system. HOXB4 transformed Rat-1 fibroblasts beyond a certain threshold level of expression. Further escalation of HOXB4 expression, however, did not enhance transformation. Instead, HOXB4 mediated a dose dependent anti-proliferative effect on Rat-1 and NIH3T3 fibroblasts. This effect was aggravated under reduced serum concentrations and was, at least partially, due to an enhanced sensitivity of HOXB4 overexpressing cells to induction of apoptosis. Based on these results we propose that HOXB4 affects cell growth in a dose-dependent manner by sensitizing cells towards extrinsic signals.