Epstein-Barr Virus (EBV) induced B cell immortalization is an efficient tool for monoclonal antibody screening and isolation. However, the low efficiency of B cell transformation by EBV limits the usage of this technique. Numerous cytokines are reported to activate and boost B cells proliferation. CD40L are also reported to provide signals to B cells that induce proliferation. In order to answer that whether cytokines and CD40L fusion protein can enhance the EBV transformation efficiency of B cells, two fusion proteins CDGM (CD40L-GM-CSF) and 4CDGM (IL-4-CD40L-GM-CSF) are designed and expression plasmids are constructed. Our results showed that both CDGM and 4CDGM can enhance B cell proliferation with higher efficiency to increase B cell proliferation in vitro. However, CDGM and 4CDGM will reduce the proliferation of B cells in the presentation of EBV infection. Both CDGM and 4CDGM could facilitate the proliferation of monocytes. Our results provide indication in B cell proliferation and optimization of EBV induced B cell immortalization.