The process of development of various cell types is often based on a linear or deterministic paradigm. This is true, for example, for osteoblast development, a process that occurs through the differentiation of a subset of primitive fibroblast progenitors called colony-forming unit-osteoblasts (CFU-Os). CFU-O differentiation has been subdivided into three stages: proliferation, extracellular matrix development and maturation, and mineralization, with characteristic changes in gene expression at each stage. Few analyses have asked whether CFU-O differentiation, or indeed stem cell differentiation in general, may follow more complex and nondeterministic paths, a possibility that may underlie the substantial number of discrepancies in published reports of progenitor cell developmental sequences. We analyzed 99 single colonies of osteoblast stem/primitive progenitor cells cultured under identical conditions. The colonies were analyzed by global amplification poly(A) polymerase chain reaction to determine which of nine genes had been expressed. We used the expression profiles to develop a statistically rigorous map of the cell fate decisions that occur during osteoprogenitor differentiation and show that different developmental routes can be taken to achieve the same end point phenotype. These routes appear to involve both developmental "dead ends" (leading to the expression of genes not correlated with osteoblast-associated genes or the mature osteoblast phenotype) and developmental flexibility (the existence of multiple gene expression routes to the same developmental end point). Our results provide new insight into the biology of primitive progenitor cell differentiation and introduce a powerful new quantitative method for stem cell lineage analysis that should be applicable to a wide variety of stem cell systems.