Objective To investigate the feasibility and effectiveness of the 96-well plate suspension method for differentiation of P19 cells into adipocytesin vitro.Methods P19 cells were cultivated in aggregates termed embryoid bodies (EBs) in the 96-well plate containing a thin layer of low-gelling temperature agarose for 7 days with the cultivation medium, supplemented with all-trans retinoic acid (RA) between the 2nd and the 5th day. The EBs were transferred into gelatin-coated culture dishes and cultivated for another 20 days in the presence of insulin and triiodothyronine (T3). Oil-Red-O staining was applied to indicate the generation of adipocytes. Results The 96-well plate suspending culture facilitated the formation of the EBs in similar size. At the end of this period, part of cells present in the EBs outgrowth formed adipocyte colonies, which were characterized by round in shape and small lipid droplets scattered throughout cytoplasm. Adipocytes were stained with Oil Red O for fat droplets. Conclusion P19 cell-derived EBs formed in 96-well plate suspension could undergo adipocyte differentiation in vitro.