The paper introduces a novel computational approach called Spanning-tree Progression Analysis of Density-normalized Events (SPADE) for analyzing high-dimensional single-cell data, particularly cytometry data. SPADE is designed to objectively uncover cellular heterogeneity and organize cells into a hierarchy of related phenotypes without requiring prior knowledge or subjective gating. The method involves density-dependent downsampling, agglomerative clustering, minimum spanning tree construction, and upsampling to map cells onto the resulting tree structure. SPADE was applied to both mouse and human bone marrow datasets, successfully recovering known hematopoietic hierarchies and identifying novel cell types, such as natural killer (NK) cells, without using specific NK markers. SPADE also allowed for the visualization of intracellular signal activation across hematopoietic development and the comparison of functional markers in response to perturbations. The authors demonstrate the robustness and scalability of SPADE through various tests and comparisons with traditional gating methods, highlighting its potential for facilitating new biological discoveries and exploring cellular heterogeneity.The paper introduces a novel computational approach called Spanning-tree Progression Analysis of Density-normalized Events (SPADE) for analyzing high-dimensional single-cell data, particularly cytometry data. SPADE is designed to objectively uncover cellular heterogeneity and organize cells into a hierarchy of related phenotypes without requiring prior knowledge or subjective gating. The method involves density-dependent downsampling, agglomerative clustering, minimum spanning tree construction, and upsampling to map cells onto the resulting tree structure. SPADE was applied to both mouse and human bone marrow datasets, successfully recovering known hematopoietic hierarchies and identifying novel cell types, such as natural killer (NK) cells, without using specific NK markers. SPADE also allowed for the visualization of intracellular signal activation across hematopoietic development and the comparison of functional markers in response to perturbations. The authors demonstrate the robustness and scalability of SPADE through various tests and comparisons with traditional gating methods, highlighting its potential for facilitating new biological discoveries and exploring cellular heterogeneity.