One characteristic of the erythroid terminal differentiation is hemoglobin expression and enucleation. In the present study, by using real-time cultured Friend Virus Anemia-inducing (FVA) cells, scanning electron microscopy (SEM), immunofluorescence combined with laser scanning confocal microscopy (LSCM), the FVA cells induced by Erythropoietin (EPO) for were used for: 1) to study the shape and proportion of cells at different differentiating stages, enucleation process, the formation of blood islands, and engulfment of nuclei by macrophages in real time, 2) to quantitatively analyze erythroid cell surface markers including CD71 and Ter119, and cytoskeletal-associated proteins (stathmin, septin8 and RBBP4). The results of real-time monitoring of enucleation indicated that it took about 7 to 8 hours to extrude the nuclei from karyopyknosis (polychromatic erythroblasts). It further showed that the macrophages engulfed the expelled erythroid nuclei. SEM showed a variety of shapes of the nascent reticulocytes. In the process of erythroid differentiation, expressiosn†of both the transferring receptors CD71 and Ter119 were higher than that of adult blood cells, whereas, cytoskeletal-associated proteins (stathmin, septin8 and RBBP4) decreased gradually. Therefore, systematic observation of the process of differentiation and enucleation will provide a deeper understanding of cellular and molecular mechanisms for erythroid differentiation and carcinogenesis.