Background: The hematologic response to hydroxyurea (HU) is varied among β-thalassemia (BT) patients. The BCL11A and SOX6 genes are involved in response to HU. This study aimed to investigate the in-vitro responsiveness of HU among BT major patients homozygote for IVSII-1G>A mutation and XmnI single nucleotide polymorphism (SNP) in order to find whether the in-vitro Hb concentration is a predictor of clinical (HU) responsiveness. Methods: In this case-control study, twenty BT patients homozygote for IVSII-1G>A mutation and XmnI SNP from Thalassemia Research Center, Sari, Iran in 2015 were selected and categorized into two groups of 10 Responder (R) and 10 Non-Responder (NR) according to their clinical HU response. Ten healthy individuals as a control group were also selected. Hematopoietic erythroid progenitors were expanded from peripheral blood. Hb concentration was measured using photometry method. The flow cytometry and real-time PCR methods were applied for the analysis of cell surface markers (CD71 and CD235a) and gene expression (BCL11A and SOX6), respectively. Results: R and NR groups produced higher amount of Basic Hb than C group in cell culture medium at day 14 (P