Albumin, the most abundant plasma protein, not only controls osmotic blood pressure, but also serves as a carrier for various small molecules, including pharmaceuticals. Its impact on pharmacological properties of many drugs has been extensively studied over decades. Here, we focus on its interaction with the following mobilizing agents: Granulocyte-colony stimulating factor (G-CSF) and AMD3100, where such analyses are lacking. These compounds are widely used for hematopoietic stem cell mobilization of healthy donors or patients. Using albumin-deficient (Alb−/−) mice, we studied the contribution of albumin to mobilization outcomes. Mobilization with the bicyclam CXCR4 antagonist AMD3100 was attenuated in Alb−/− mice compared to wild-type littermates. By contrast, mobilization with recombinant human G-CSF (rhG-CSF), administered twice daily over a five-day course, was significantly increased in Alb−/− mice. In terms of a mechanism, we show that rhG-CSF bioavailability in the bone marrow is significantly improved in Alb−/− mice, compared to wild-type (WT) littermates, where rhG-CSF levels dramatically drop within a few hours of the injection. These observations likely explain the favorable mobilization outcomes with split-dose versus single-dose administration of rhG-CSF to healthy donors.