Several types of peripheral blood cells express ABC transporters. ABCC4 (MRP4) and ABCC5 (MRP5) localize to different cellular sites and fulfill lineage‐specific functions such as mediator storage in platelets' dense granules. All mature blood cells originate from the same precursor and specific functionalities arise during differentiation. To characterize this process, expression, localization and function of MRP4 and MRP5 were assessed in differentiating human CD34+ progenitors and leukemia cell lines using real time polymerase chain reaction (PCR), immunofluorescence microscopy and cell viability assays. Median MRP4 mRNA copy numbers were significantly enhanced by megakaryocytic differentiation from 7.9 × 10³ to 5.8 × 10⁴ copies per nanograms of total RNA (p < 0.05) in CD34+ progenitors and in M‐07e cells (MRP4 mRNA/18S rRNA ratios: 5.4 ± 3.8 × 10⁻⁴ vs. 2.7 ± 0.9 × 10⁻³ for native and differentiated cells, respectively, p < 0.05), and MRP4 protein was localized to granular structures and to the plasma membrane both in differentiated progenitors and bone marrow megakaryocytes. In contrast, expression of MRP4 decreased during maturation to leukocytes (MRP4 mRNA/18S rRNA ratios: 5.2 × 10⁻³ for native vs. 3.5 × 10⁻³ for CD34+ cells in the presence of G‐CSF, p < 0.05) and was significantly reduced in mature monocytes and granulocytes compared with progenitors (MRP4 mRNA/18S rRNA ratios: 8.1 ± 5.4 × 10⁻⁵ and 2.8 ± 1.6 × 10⁻⁴ vs. 1.2 ± 0.7 × 10⁻³, respectively, p < 0.05). Expression of MRP5 was not significantly altered under all differentiation conditions. These results indicate that MRP4 expression is differentially regulated during hematopoiesis. The increase of MRP4 together with its specific localization during differentiation toward megakaryocytes supports the concept of platelet specific functions whereas decreased transporter expression in leukocyte differentiation may have implications for chemotherapy. © 2008 Wiley‐Liss, Inc.