It has been shown that Notch signaling mediated by ligands of both Jagged and Delta families expands the hematopoietic stem cell compartment while blocking or delaying terminal myeloid differentiation. Here we show that Delta1‐ and Jagged1‐expressing stromal cells have distinct effects on the clonogenic and differentiation capacities of human CD34⁺ CD38⁺ cells. Jagged1 increases the number of bipotent [colony‐forming unit‐granulocyte macrophage (CFU‐GM) and unipotent progenitors (CFU‐granulocytes and CFU‐macrophages), without quantitatively affecting terminal cell differentiation, whereas Delta1 reduces the number of CFU‐GM and differentiated monocytic cells. Expression analysis of genes coding for Notch receptors, Notch targets, and Notch signaling modulators in supernatant CD34⁺ cells arising upon contact with Jagged1 and Delta1 shows dynamic and differential gene expression profiles over time. At early time points, modest upregulation of Notch1, Notch3, and Hes1 was observed in Jagged1‐CD34⁺ cells, whereas those in contact with Delta1 strikingly upregulated Notch3 and Hes1. Later, myeloid progenitors with strong clonogenic potential emerging upon contact with Jagged1 upregulated Notch1 and Deltex and downregulated Notch signaling modulators, whereas T/NK progenitors originated by Delta1 strikingly upregulated Notch3 and Deltex and, to a lesser extent, Hes1, Lunatic Fringe, and Numb. Together, the data unravel previously unrecognized expression patterns of Notch signaling‐related genes in CD34⁺ CD38⁺ cells as they develop in Jagged1‐ or Delta1‐stromal cell environments, which appear to reflect sequential maturational stages of CD34⁺ cells into distinct cell lineages.