This paper documents the budding and division process of human erythrocytes in vitro using time-lapse light microscopy of hanging-drop preparations. The erythrocytes were prepared from normal adult human peripheral blood. Red blood cells showed cytoplasmic budding, segmentation, and direct division with delayed addition of erythropoietin in serum-free Dulbecco's modified Eagle's medium/nutrient mixture F-12 Ham. These observations are thought to be useful for developing model of definitive erythropoiesis and simple expansion of human erythrocytes.