This chapter describes a novel cell fusion method for direct cytoplasmic transfer between live single cells. In order to realize this, we developed a microfluidic device which can make single cells pair at the position of microslit. Cell fusion was induced between the paired cells through a microslit by Sendai virus envelope (HVJ-E) to make strictured cytoplasmic connection. In the fused cell pairs, the nuclei in spread shape cells did not pass through the microslit. On the other hand, the nuclei in round shape cells migrated to the fusion partner leading to nuclear mixing. Supplementation with Y-27632, a ROCK inhibitor, prevented nuclear mixing and enhanced cell fusion rate. Mitochondria were transferred through the microslit under a condition of with or without Y-27632 supplementation. These data suggest that our cell fusion method using a newly developed microfluidic device can perform direct cytoplasmic transfer between live single cells.