Triple-negative breast cancer (TNBC) which is characterized by the lack of estrogen and progesterone receptors, and without amplification of ERBB2 gene, is highly aggressive with poor clinical outcome. Cancer stem cells (CSCs) which can promote recurrence and drug resistance of tumors are highly enriched in TNBC. Previous studies have revealed that different isoforms of Akt (Akt1-3) play distinct roles in regulating breast cancer progression. Akt1 isoform suppresses breast cancer cell invasion and migration. In contrast, Akt2 has been shown to promote motility in breast cancer cell. Akt3 is an oncogene overexpressed in TNBC. It is essential in enhancing TNBC growth and mediating resistance of breast cancer cells to Akt pathway inhibitors. However, the functional role of Akt3 in TNBC CSCs remains poorly understood. In addition, an Akt3-specific substrate awaits to be identified. We used Crispr/cas9 technique to knock out Akt (Akt1-3) gene in TNBC cell lines including MDA-MB-231 and MCF10-DCIS. Immunoblotting demonstrated efficient knockout of Akt (Akt1-3) in these cell lines. Knockout of Akt3 decreases mammosphere forming ability in both MDA-MB-231 and MCF10-DCIS cell lines in vitro. Our data with single cell clones also demonstrated that knockout of Akt3 decreases the population of ALDH+ and CD44+/CD24- cells. Furthermore, knockout of Akt3 reduces tumor initiating ability both in vivo and in vitro, indicating that Akt3 is required for tumor initiation in TNBC. This prompted us to investigate the molecular mechanisms by which Akt3 regulates TNBC stemness. By performing RT-PCR, a total of 30 CSC markers/regulators were selected to identify the potential target genes of Akt3. Knocking out Akt3 significantly decreases Snail/Slug expression accompanied with suppressed CSC properties, whereas overexpression of Akt3 promotes stemness phenotypes. Through analyzing clinical datasets, we showed that Akt3 expression in breast cancer is correlated with Snail and Slug expression as well as pYB1 Ser102 levels. In addition, high expression of YB1 is associated with poor prognosis in breast cancer patients. Our data demonstrated that overexpression of YB1 promotes TNBC stemness whereas depleting YB1 expression has the opposite effect. Mechanistically, Akt3, but not Akt1 or Akt2, specifically phosphorylates YB1 at Ser102 in TNBC cells and in in vitro kinase assay. Overexpression of YB1 rescues mammosphere formation ability in Akt3-knockout cells, suggesting that YB1 is a downstream molecule of Akt3 in mediating TNBC stemness. To the best of our knowledge, our findings identified YB1 as the first bona fide Akt3-specific substrate. Importantly, phosphorylation of YB1 at Ser102 by Akt3 plays a critical role in the self-renewal of TNBC CSCs, suggesting that disrupting the Akt3-YB1 signaling axis might provide new therapeutic opportunity for TNBC. In addition to Akt3-YB1 axis, we investigated a novel role of a ribosome biogenesis gene NIP7 in the tumorigenesis of TNBC. Using TCGA datasets, we found that NIP7 mRNA is overexpressed in 26% of TNBCs, compared to 3% in luminal breast tumors. Little is known about the functional role of NIP7 in human cancer. By analyzing BCIP dataset, we found that overexpression of NIP7 associates with shorter survival of TNBC patients especially for Stage I and Stage II TNBC patients. By using Crispr/Cas9-mediated inducible knockout of NIP7, we showed that NIP7 depletion inhibits TNBC cell proliferation in both 2D and 3D cultures. NIP7 knockout also significantly inhibits the progeny producing capability of TNBC cells as shown by clonogenic assays. NIP7 knockout also results in cell cycle arrest and increased apoptosis of TNBC cells. By performing mammosphere assay, we further demonstrated the critical role of NIP7 in CSC phenotypes in vitro. NIP7 knockout impairs migration with enhanced E-cadherin expression and reduced N-cadherin and vimentin expression, in line with its potential role in driving epithelial-to-mesenchymal transition (EMT). Importantly, we have performed preclinical studies and showed that NIP7 knockout significantly inhibits HCC1806 and BT549 TNBC xenograft growth. NIP7 knockout potently reduces expression of 5s and 45s rRNA as well as 28s rRNA. By analyzing Depmap, we found that UTP4 is the top co-dependency gene of NIP7 in tumor cells. It has been shown previously that knockdown of UPT4 in colorectal cancer cells reduced colony formation significantly. Our data demonstrated that NIP7 knockout leads in reduced UTP4 protein expression without affecting its mRNA expression, suggesting that NIP7 regulates UTP4 expression at the translational or post-translational level. Taken together, our findings identify NIP7 as a novel cancer-related gene promoting TNBC growth and stemness properties. Targeting NIP7 may provide a new strategy for TNBC treatment.