Larp4b is a member of the LARP family, which can interact with RNA and generally stimulate the translation of mRNA. Abnormal expression of Larp4b can be found in leukemia patients in our previous study. This study was purposed to detect the relative expression of Larp4b mRNA in different subpopulations of mouse hematopoietic cells, to construct lentivirus vector containing shLarp4b targeting mouse gene Larp4b and to explore its effects on mouse Lin(-) cells infected with shLarp4b by lentivirus. SF-LV-shLarP4b-EGFP and control vectors were constructed and two-plasmid lentivirus packing system was used to transfect 293T cells. After 48 h and 72 h, lentivirus SF-LV-shLarp4b-EGFP was harvested and was used to infect Lin(-) cells. After 48 h, EGFP(+) cells was sorted by flow cytometry (FCM). Meanwhile, semi-quantitative real time-PCR, AnnexinV-PE/7-AAD staining, PI staining and colony forming cell assay (CFC) were performed to determine the expression of Larp4b and its effect on the proliferation of hematopoietic progenitor cells. The results showed that Larp4b was highly expressed in myeloid cells. SF-LV-shLarp4b-EGFP was successfully constructed according to the restriction endonuclease digestion assay. RT-PCR confirmed that Larp4b was efficiently knockdown in mouse Lin(-) cells. The low expression of Larp4b did not affect the colony forming number, the apoptosis and cell cycle of Lin(-) cells. It is concluded that knockdown of Larp4b in mouse Lin(-) cells do not contribute to the colony forming ability and the growth of Lin(-) cells in vitro. This useful knockdown system will be used to study in vivo Larp4b in future.