Background: Donor-derived red blood cells (RBCs) are the most common form of cellular therapy, coupled with donor-dependency, alloimmunization and blood borne disease risks. In vitro derivation of RBCs from an immortal source, as iPSCs provide an alternative solution to these issues. In addition, in vitro iPSC-RBCs allow genome editing for possible gene therapies, while large scale production would be beneficial in RBC drug delivery. The current limitation of iPSC-RBCs is their development immaturity, leading to low enucleation potential, which requires improvement prior clinical application. Aims: We aim to push iPSC-derived erythroid differentiation to definitive erythropoiesis yielding enucleated erythrocytes Methods: A minimum of three independent iPSC lines were used during the studies. These iPSC lines were single cell seeded and emerging colonies were differentiated towards erythroblasts using specific growth factor cocktails and media developed at Sanquin. Human mononuclear cells isolated from fetal liver, cord blood and adult blood were also differentiated to erythroblasts. Flow cytometry was used to assess culture purity, stage of erythropoiesis and enculeation. RT-PCR and HPLC was used to quantify the developmentally defined globins. RNA was isolated from the different erythroblasts and RNA-sequencing was performed and analysed. Single cell RNA-seq was performed on single cell FACS-sorted iPSC derived hematopoietic cells. Results: To further develop the process, we have established a GMP-compatible iPSC generation and differentiation protocol, that upon hematopoietic specification yielded erythroblasts (EBL). This method resulted in 2–6∗105 EBLs/iPSC with 100% CD71+/CD235+ (erythroid-specific markers). Further maturation yielded orthochromatic normoblasts containing a mix of primitive and definitive erythroid waves, leading to poor enucleation potential and/or reticulocyte stability. Total RNAseq analysis comparing iPSC to in vitro derived definitive erythroid cells from fetal liver, cord and adult revealed significant differences and identified specific genes that may be used to further augment iPSC-erythroid terminal differentiation to erythrocytes. For instance, iPSC-derived erythroblasts lack expression of specific erythroid regulators (e.g. MYB and KLF1 target genes SOX6, BCL11A and BCAM). Single-cell RNAseq of early iPSC differentiation was performed, aiming to recognize distinct waves of hematopoiesis. Five cell clusters were identified of which, one was HSPC-like, 3 lineage-committed populations and 1 of unknown origin. This data coupled with index sorting, defined the HSPCs as CD71+/CD235−, and the erythroid cells as CD71+/CD235+ expressing embryonic and fetal Hbs. In addition, we have found differentially expressed markers between the iPSC-RBCs and definitive erythroid cells that may be used to better define specific erythroid waves during iPSC differentiations and/or fetal development. We hypothesize, that the identified HSPCs potentially give rise to developmentally more mature cells, which we aim to expand. Currently we are pursuing different approaches among which forward programming, to support early iPSC-HSPCs that produce developmentally more mature definitive erythroid cells including proper enucleation potential. Summary/Conclusion: In conclusion, we have performed extensive RNA analysis of iPSC, cord, fetal and adult erythroblast and identified potential targets that may aid in proper iPSC-derived erythroid differentiation to enucleated red cells.