Our previous study showed that Hydrogen Sulfide (H2S) could significantly increase the platelet number in mice with radiation-induced myelosuppression but the specific mechanism had not been elucidated. The aim of this study was to explore the effect of H2S on megakaryocytes (MKs) differentiation and apoptosis, then speculate the role of H2S in Thrombopoiesis. Here we selected MEG01 and DAMI cell lines as the research objects. Then the effects of H2S on MK differentiation and apoptosis were comprehensively explored from various aspects such as the changes of cell surface markers, cell cycle, cell apoptosis, protein phosphorylation of signaling pathways, transcriptome difference and enrichment analysis as well as transcription factor changes, in order to find out whether H2S can promotes platelet release by affecting MKs differentiation and apoptosis. Results showed that H2S could promote the transformation of MKs cell cycle from G0/G1 phase to S phase, which was conductive to DNA replication and promoted MKs apoptosis, and it’s pro-apoptosis role on MEG01 is depended on Caspase 3; H2S could significantly increase the expression of GATA-1 and Fli-1, which are related to MKs differentiation and mitosis, and H2S could increase the expression of NF-E2, which is related to platelet-formation. Altogether, our finding indicate that H2S can promote MKs differentiation and apoptosis, which are related to platelet-formation.