Pretreatment with Tmprss6-ASO followed by a concomitant lower dose of EPO is more effective than Tmprss6-ASO alone in improving anemia in Hbb^th3/+ mice. Thalassemic mice receiving cotherapy with Tmprss6-ASO (T6-ASO) and lower dose of EPO (in the form of rhEPO or fibroblasts overexpressing Epo) following a pretreatment with Tmprss6-ASO showed significantly improved anemia when compared with animals receiving Tmprss6-ASO alone. At the end of the treatment we observed increased RBC number, Hb levels and reduced splenomegaly when both a low dose of rhEPO (A-C) or lower dose of fibroblasts overexpressing Epo (D-F) were coadministered with Tmprss6-ASO. Dotted red line indicates mean values for untreated WT mice. Dashed black lines indicates mean values for untreated Hbb^th3/+ mice. Bars represent SD. Asterisks refer to statistically significant differences (****P ≤ .001; ***P ≤ .005; **P ≤ .01; *P ≤ .05).