Fms-like tyrosine kinase (Flt3L) is a potent stimulator of hematopoietic progenitor cell (HPC) expansion and mobilization; however, this requires 7–10 days of administration. We investigated whether sustained delivery of Flt3L using a poloxamer-based matrix (PG) could accelerate and/or improve the hematopoietic activity of Flt3L in mice. A single injection of PG-Flt3L stimulated significantly more rapid and greater HPC mobilization to the spleen and peripheral blood than the daily injection of Flt3L formulated in saline. Pharmacokinetic analysis demonstrated that the formulation of Flt3L in PG prolonged its elimination (Tβ) half-life (2.3-fold) and increased its bioavailability (> two fold) and the time to maximum serum concentration (T max)(2.7-fold). Further, coadministration of G-CSF and PG-Flt3L allowed lower doses of Flt3L to be active, with significantly greater hematopoietic and mobilization activity, compared to the same total dose of G-CSF, Flt3L or G-CSF and Flt3L formulated in saline. These data demonstrate that formulation of Flt3L in PG significantly accelerates and increases HPC expansion and mobilization. The observation of increased bioactivity by PG-Flt3L in rodents suggests the potential for improved clinical efficacy of Flt3L by reducing the time required for HPC mobilization.