HIV-derived lentiviral vectors have been used widely to transduce non-dividing cells, such as hematopoietic stem cells (HSCs), in the setting of gene therapy. In this study, we screened lentiviral vectors for their ability to drive expression of the murine MHC class II chaperone, invariant chain (Ii) and a GFP reporter. The vectors included T2A vector with T2A-separated Ii and GFP under the same MSCV promoter, dual-promoter vectors with separate promoters for Ii and GFP (called MSCV or EF1a according to the promoter driving Ii expression), and a vector with EF1a driving a fusion of Ii/GFP (called Fusion vector). T2A and MSCV induced the highest levels of Ii and GFP expression, respectively, after direct transfection of 293T cells. All vectors except the Fusion vector drove expression of functional Ii, based on the enhancement of MHC class II level, which is a known consequence of Ii expression. Comparing the vectors after they were packaged into lentiviruses and used to transduce 293T, we found that MSCV and EF1a vectors mediated higher Ii and GFP expression. In ckit(+) bone marrow (BM) cells, MSCV still induced the highest Ii and GFP expression, whereas EF1a induced only robust Ii expression. Regardless of the vector, both Ii and GFP levels were significantly reduced in BM cells compared to 293T cells. When in vivo expression was assessed in cells derived from MSCV-transduced BM-HSCs, up to 80% of myeloid cells were GFP(+), but no Ii expression was observed. In contrast, transplantation of EF1a-transduced BM-HSCs led to much higher in vivo Ii expression. Thus, among those compared, dual-promoter vector-based lentivirus with the EF1a promoter driving the gene of interest is optimal for murine BM-HSC transduction.