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Patients with homozygous beta thalassemia and the β⁰β⁰ genotype produce almost only fetal hemoglobin and a small proportion of hemoglobin A2. However, in the bone marrow of these patients there are descriptions of two different subpopulations of erythroblasts, one with high production of HbF (High HbF cells) and another with low production of HbF (Low HbF cells). The High F cells are probably those that survive through erythropoiesis and originate the viable red blood cells. The molecular mechanisms that are responsible for this high production of HbF are not clear. Therefore, the central aim of our work was to study, in cultures of CD34⁺ cells from β⁰β⁰ patients, differentiated to erythrocytes in vitro, the molecular characteristics of cells related to high production of HbF (High HbF), compared to those with low production of HbF (Low HbF). For this purpose, peripheral blood was collected from patients and CD34⁺ cells were identified, and further differentiated in vitroand then isolated by flow cytometry (FACS Aria). After separating the High HbF and Low HbF groups, the cells were morphologically evaluated by optical and confocal microscopy and image cytometry. Next RNA was extracted from the pool of isolated cells and the RNA sequencing assay (RNAseq) was performed, so that it was possible to analyze the transcripts that were differentially expressed between each of the groups. Finally, we validated the sequencing data by real-time PCR. The sorting was performed on the 10th day of cell culture, when the cells presented a double labeling profile for anti-transferrin (CD71) and anti-glycophorin (CD235) antibodies, the mean intensity and fluorescence (MIF) of the markers was 1058 and 1365 for CD71 in healthy control and β-thalassemia groups, respectively, and for CD235 was 430 in controls and 516 for patients. The cells were in an intermediate stage of maturation. After the isolation of sub-populations of High HbF and Low HbF cells, confocal microscopy indicated a clear difference in the intracellular HbF levels of the analyzed cells, confirming that the sorting by cytometry had occurred as desired. Imaging cytometry analysis revealed a MIF of 7.8 x 10⁵ for High HbF cells and 1.7 x 10⁵ for Low HbF cells, when we assessed the cell pools and when single cells were studied, these values ranged from 2.2 x 10⁵ for Low HbF cells to 1.9 x 10⁶ of mean intensity and fluorescence for High cells. After all data generated by RNAseq were filtered, we obtained a list of 16 enriched and differentially-expressed genes between High HbF and Low HbF cells. From these genes, it was possible to identify two that were apparently associated with the increased production of fetal hemoglobin, the genes ZBTB8Band LIN28B. ZBTB8Bis a protein-coding gene with a zinc finger domain and may be involved in transcriptional regulation, it was included in our analyses since this gene is part of the same family as ZBTB7A, an important transcription factor that represses several genes involved in cell differentiation and proliferation and that has already been described as a regulator of HbF production. The LIN28Bgene encodes an RNA-binding protein, which is described as a possible regulator of HbF levels during fetal development, by increasing the expression of gamma globin, culminating in the increase of intracellular levels of fetal hemoglobin through a direct action on the let-7 microRNA family and BCL11A gene.These results not only contribute to a better understanding of the mechanisms of gene regulation involved in the production of HbF, but indicate a potential new therapeutic approach to increase the production of HbF in hemoglobinopathies.