Stem cell differentiation must be regulated by intricate and complex interactions between cells and their surrounding environment, ensuring normal organ and tissue morphology such as the liver1. Though it is well acknowledged that microgravity provides necessary mechanical force signals for cell fate2, how microgravity affects growth, differentiation, and communication is still largely unknown due to the lack of real experimental scenarios and reproducibility tools. Here, Rotating Flat Chamber (RFC) was used to simulate ground-based microgravity effects to study how microgravity effects affect the differentiation of HepaRG (hepatic progenitor cells) cells. Unexpectedly, the results show that RFC conditions could promote HepaRG cell differentiation which exhibited increased expression of Alpha-fetoprotein (AFP), albumin (ALB), and Recombinant Cytokeratin 18 (CK18). Through screening a series of mechanical receptors, the ion channel TRPML1 was critical for promoting the differentiation effect under RFC conditions. Once TRPML1 was activated by stimulated microgravity effects, the concentration of lysosomal calcium ions was increased to activate the Wnt/beta-catenin signaling pathway, which finally led to enhanced cell differentiation of HepaRG cells. In addition, the cytoskeleton was remodeled under RFC conditions to influence the expression of PI (3,5) P2, which is the best-known activator of TRPML1. In summary, our findings have established a mechanism by which simulated microgravity promotes the differentiation of HepaRG cells through the TRPML1 signaling pathway, which provides a potential target for the regulation of hepatic stem/progenitor cell differentiation and embryonic liver development under real microgravity conditions.