Glycosylated antitumor ether lipids (GAELs) 6 and 7 containing a α- or β-D-gluco-configured 2-amino-2-deoxy (2-NH2-Glc) sugar moiety linked to a glycerolipid aglycone kill cancer cell lines via a non-apoptotic mechanism that could be exploited to kill cancer stem cells. To test this hypothesis and develop novel potent GAEL analogs, we synthesized GAELS which contain D-galacto- and D-manno-configured 2-amino-2-deoxy sugar moieties (2-NH2-Gal or 2-NH2-Man) and investigated their cytotoxicity against human epithelial cancer cell lines and cancer stem cells derived from BT-474 breast cancer cells. Within the class of D-galacto-configured GAELs, we prepared both O- and S-glycosidic linkages as well as their corresponding α- and β-anomers and screened against breast (BT-474, JIMT-1 and BT-549), pancreas (MiaPaCa2) and prostate cancer (DU145, PC3) cancer cell lines. The α-anomeric 2-NH2-Gal-based lipid 1 was the most active of all the compounds tested with CC50 values of 4.4-8 μM and is the most active GAEL synthesized to date. The β-anomer 2 was 4->5-fold less active than 1. Replacement of the α-O-glycosidic by an α-S-glycosidic linkage resulted in a 2-4-fold reduction in activity, while the β-S-glycolipid 4 was inactive. In comparison, α-configured 2-NH2-Man-based glycerolipid 5 displayed very little activity with CC50 > 30 μM. The effect of the most active GAELs, 1, 6, or 7, on cancer stem cell viability revealed that all three inhibited the formation of tumorspheres from BT-474 cancer stem cell lines, caused the disintegration of preformed tumorspheres and resulted in total loss of cell viability of the cancer stem cells at concentrations of 20 μM. In contrast, the related antitumor ether lipid gold standard, edelfosine that is in clinical development was much less effective in preventing tumorsphere formation and affecting the viability of the cancer stem cells. Taken together our study demonstrates that α-GAEL anomers are more potent than their corresponding β-anomers and that the nature of the CHO moiety as well as the glycosidic bond significantly affects activity. The study also showed that GAELs are effective in killing CSCs while the apoptosis-inducing edelfosine is not.