Cancer immunotherapy has made a big advance on benefit for cure the patients from malignancy. Recent hypothesis indicates that the crosstalk between cancer cells and tumor microenvironment can regulate the innate and adaptive immune system drives immunosuppressive condition for the immune escape of cancer cells. Using genome-wide microarray and RNA-sequencing analysis, the immune-related gene profiling has been identified in the lung cancer stem cells (CSCs). The CSCs-condition medium was collected from the CSCs or CSCs/CAFs co-culture system to treat and analyze the functional responses of the tumor-associated macrophage (TAMs)/M2 macrophage and T cells populations. The flow cytometry and Q-PCR were used to validate the markers and related gene expression. We found that the tumor microenvironment is crucial to support the tumor malignancy and cancer stemness through paracrine networks (e.g., IGFII, HGF, LIF, IL-4/6/17, BMP-1/2, and CXCL6); as well as, the niche showed the impacts on the immune regulation. According to the genome-wide microarray and RNA-sequencing transcriptomic analysis of the lung cancer stem cells (CSCs), we have identified a cluster of genes and signaling involved in the immune modulation under the tumor microenvironment. Such CSCs-condition medium could significantly induce the interferon signaling, and immune checkpoints, CD274 and CD273 (PDL1 and PDL2), expression in CSCs. The population of tumor-associated macrophage (M2 population) from the malignant pleural effusion could be maintained via the CSCs-condition medium concentration-dependently. Most importantly, the population of the regulatory T cells (Treg) and the TAMs could be increased via the condition medium from CSCs; whereas, the CD8+ T-cells was significantly reduced. According to the transcriptomic/proteomic analysis of the CSCs and the niche, we have identified several important immune modulating signaling; affecting the paracrine and cytosine networks, which modulate the T cells population and may correlate with the immune escape of lung CSCs. Our results have identified the roles of CSCs on the immune modulation in the tumorous microenvironment, targeting on CSCs and related immune modulating signaling could be benefit for improving the antigen presentation and inhibit the immune checkpoints PDL1 and PDL2 expression in the combinatory anti-cancer immunotherapy.