Abstract Tumor initiating cells (TIC) foster treatment resistance and tumor relapse. We show here that in mouse B16 (melanoma) and ID8agg (ovarian carcinoma), and human ovarian cancer ES2, PD-L1 knockdown (shRNA) reduced TIC numbers and functions. Data shown are from 2 PD-L1lo clones/cell type. Sorted, control CD44+CD133+CD24+ (B16), CD44+CD24+ (ID8agg) and ALDHhi (ES2) TIC formed 50-75% more and bigger spheres vs. PD-L1lo TIC in vitro, consistent with reduced TIC self-renewal in PD-L1lo. In vivo, PD-L1lo B16 and ID8agg TIC exhibited reduced tumorigenicity in wild type mice, consistent with poor TIC function, but immune effects could not be excluded. B16 and ES2 PD-L1lo TIC had significantly reduced tumorigenicity versus control TIC in immune deficient NSG mice consistent with cell-autonomous, immune-independent TIC promotion by PD-L1. In further confirmation, we found reduced serial transplantability of PD-L1lo B16 TIC in NSG mice. The canonical stemness genes nanog and pou5f1 (oct4) were significantly higher in control vs. PD-L1lo TIC for B16 and ID8agg. The canonical stemness gene SOX2 was significantly higher in control vs. PD-L1lo ES2 TIC. Altogether, these data validate effects in human cells, and are consistent with PD-L1 driving TIC through stemness gene control, although specific genes differed in distinct tumors (likely due to differing mutational landscapes). mTORC1 signaling linked to stemness (assessed by raptor) was lower but mTORC2 was unaffected (rictor) in PD-L1lo vs control TIC from all 3 tumors, suggesting mTORC1 control of PD-L1-mediated TIC generation. In support, the mTORC1 inhibitor rapamycin reduced control TIC significantly (>50% vs. untreated). Strikingly, rapamycin significantly increased PD-L1lo TIC in all 3 tumors, suggesting mTORC1 effects differ by tumor PD-L1 status. Interferon (IFN)-γ similarly reduced control TIC and augmented PD-L1lo TIC numbers, suggesting the novel concept that PD-L1 alters IFN-γ signals. PD-L1 sensitization of TIC to IFN-γ and rapamycin is the first example of a molecule enhancing TIC treatment response to our knowledge. We recently reported that αPD-L1 directly reduces B16 and ID8agg proliferation in vitro and in vivo (accession no. 27671674). Preliminary data now show that αPD-L1 (and αPD-1) directly reduce TIC proliferation, and these effects differ from non-TIC. We challenge the paradigm that tumor PD-L1 is primarily an immune escape molecule and that αPD-L1 works primarily to block T cell PD-1 interactions. Citation Format: Harshita B. Gupta, Curtis A. Clark, Bin Yuan, Gangadhara Sareddy, Srilakshmi Pandeswara, Alvaro S. Padron, Vincent Hurez, Jose-Conejo Garcia, Ratna Vadlamudi, Rong Li, Tyler J. Curiel. Cell-intrinsic PD-L1 directs tumor initiating cell fate and rapamycin and interferon-γ response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5649. doi:10.1158/1538-7445.AM2017-5649