Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia in which 25% of the patients have a mutation in the ribosomal protein S19 (RPS19) gene. It is unknown how the ribosomal protein deficiency leads to anemia. We previously developed three lentiviral vectors expressing siRNA against RPS19 and one scramble control vector. All vectors also express GFP. We have previously shown that transduction of CD34+ bone marrow (BM) cells with the siRNA vectors reduced RPS19 mRNA levels, resulting in formation of fewer erythroid colonies. In the present study, we have demonstrated downregulation of RPS19 protein in siRNA treated cells. RPS19 protein levels decreased over time and were reduced to 40-60% of normal in cells transduced with all three siRNA vectors 5 days after transduction. We asked which stage of erythroid development is most affected by RPS19 deficiency. After 7 days in liquid culture supporting erythroid differentiation Glycophorin A (GlyA) and CD71 expression was examined by FACS. RPS19-silenced as well as DBA patient CD34+ cells exhibited a block in erythroid differentiation seen as an increased fraction of GlyAneg CD71low cells while the fractions of CD71high GlyAintermediate and GlyAhigh cells decreased. We cultured untransduced CD34+ cells in liquid culture for 7 days and isolated CD71high GlyA intermediate cells that are highly enriched in CFU-E and do not contain BFU-E. These cells were transduced with RPS19 siRNA vectors. Further erythroid maturation from CFU-E (CD71high GlyAintermediate) to more mature erythroid cells (GlyAhigh) was not affected by RPS19 silencing suggesting that the main block in erythroid development occurs prior to the CFU-E formation. The failure of erythroid differentiation correlated with the decrease in RPS19 protein levels. Transduction with a lentivirus expressing an siRNA-resistant RPS19 transcript rescued both the erythroid progenitor proliferation and differentiation, showing that the DBA-like phenotype is specific to the RPS19 deficiency. Finally we cultured the cells in liquid medium supporting both erythroid and myeloid differentiation. Proliferation decreased while the ratio of mature myeloid to erythroid cells increased 3 fold in cells transduced with the 2 most efficient siRNA-vectors, meaning that erythroid development is more sensitive to low RPS19 levels than myeloid development. When RPS19 is downregulated to intermediate levels, erythroid differentiation and proliferation of erythroid progenitors is severely reduced. More severe reduction of RPS19 impairs proliferation of myeloid progenitors as well, leading to a reduced output of myeloid progeny. Although our data cannot rule out hypothetical extraribosomal mechanisms we suggest that the major clinical findings in RPS19 deficient DBA can be explained by insufficient protein translation. This study shows for the first time that RPS19 protein downregulation decreases the proliferative capacity of hematopoietic progenitors leading to an early defect in erythroid development.